A83543 compounds and process for production thereof

ABSTRACT

New A83543 components, including fermentation products A83543K, A835430, A83543P, A83543U, A83543V, A83543W and A83543Y and N-demethyl derivatives, and salts thereof, are useful for the control of insects and mites. The pseudoaglycones of the new A83543 components are useful for the preparation of A83543 components. Methods are provided for making the new A83543 components by culturing of Saccharopolyspora spinosa NRRL 18395, NRRL 18537, NRRL 18538, or NRRL 18539, or NRRL 18743 or NRRL 18719 or NRRL 18823 in suitable culture medium. Insecticidal and ectoparasiticidal compositions containing new A83543 components are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

This is a divisional of application Ser. No. 08/030,522 filed Mar. 12,1993 now abandoned.

FIELD OF THE INVENTION

The invention relates to new components of fermentation product A83543.

BACKGROUND OF THE INVENTION

Target insects are rapidly developing resistance to the insecticideswhich are presently available. Resistance to insecticides in arthropodsis widespread, with at least 400 species exhibiting resistance to one ormore insecticides. The development of resistance to older insecticides,such as DDT, the carbamates, and the organophosphates, is welldocumented (see Brattsten, et al. (1986), Science, 231:1255). Resistanceto synthetic insecticides has developed extremely rapidly, including thedevelopment of resistance to the newer pyrethroid insecticides (seePickett (1988), Chem. Britain, 137). Therefore, new insecticides are indemand.

Fermentation product A83543, a family of related compounds produced bySaccharopolyspora spinosa, was recently discovered and was shown toexhibit excellent insecticidal activity. A83543 and its individualcompounds are useful for the control of mites and insects, particularlyLepidoptera and Diptera species.

By "A83543 compounds" is meant components consisting of a 5,6,5-tricylicring system, fused to a 12-membered macrocyclic lactone, a neutral sugarand an amino sugar (see Kirst et al. (1991), Tetrahedron Letters,32:4839). The family of natural components of A83543 include a genustaught in EPO Application No. 0375316 and having the following generalformula: ##STR1## wherein R¹ is H or a group selected from ##STR2## andR², R⁴, R³, R⁵ and R⁶ are hydrogen or methyl; or an acid addition saltthereof when R¹ is other than hydrogen.

The family of compounds from A83543 fermentation product has been shownto comprise individual components A83543A, A83543B, A83543C, A83543D,A83543E, A83543F, A83543G, A83543H and A83543J (see European PatentPublication No. 0 375 316); individual components A83543L, A83543M andA83543N (see copending U.S. patent application Ser. No. 07/790,287,filed Nov. 8, 1991); and individual components A83543Q, A83543R, A83543Sand A83543T (see the copending United States Patent Application ofTurner, Broughton, Huber and Mynderse, entitled "New A83543 Compoundsand Processes for Production Thereof" (U.S. patent application Ser. No.07/973,121), filed on Nov. 6, 1992). The structures of these individualcomponents and pseudoaglycones derived therefrom are shown below.##STR3## wherein R¹, R², R³, R⁴, R⁵ and R⁶ are for each component asfollows:

    ______________________________________                                        Structures of A83543 Components                                               Component  R.sup.1                                                                             R.sup.2  R.sup.3                                                                           R.sup.4                                                                              R.sup.5                                                                           R.sup.6                              ______________________________________                                        A          (a)   Me       H   Me     Me  Me                                   B          (b)   Me       H   Me     Me  Me                                   C          (c)   Me       H   Me     Me  Me                                   D          (a)   Me       Me  Me     Me  Me                                   E          (a)   Me       H   H      Me  Me                                   F          (a)   H        H   Me     Me  Me                                   G          (d)   Me       H   Me     Me  Me                                   H          (a)   Me       H   Me     H   Me                                   J          (a)   Me       H   Me     Me  H                                    L          (a)   Me       Me  Me     Me  H                                    M          (b)   Me       H   Me     Me  H                                    N          (b)   Me       Me  Me     Me  H                                    Q          (a)   Me       Me  Me     H   Me                                   R          (b)   Me       H   Me     H   Me                                   S          (a)   Me       H   H      H   Me                                   T          (a)   Me       H   Me     H   H                                    PsaA1      H     Me       H   Me     Me  Me                                   PsaD1      H     Me       Me  Me     Me  Me                                   PsaE1      H     Me       H   H      Me  Me                                   PsaF1      H     H        H   Me     Me  Me                                   PsaH1      H     Me       H   Me     H   Me                                   PsaJ1      H     Me       H   Me     Me  H                                    PsaL1      H     Me       Me  Me     Me  H                                    PsaQ1      H     Me       Me  Me     H   Me                                   PsaR1      H     Me       H   Me     H   Me                                   PsaS1      H     Me       H   H      H   Me                                   PsaT1      H     Me       H   Me     H   H                                    ______________________________________                                    

Sinefungin, an antibiotic of microbial origin, has been shown to inhibitspecific S-adenosylmethionine-dependent methyltransferases. Thiscompound is effective in inhibiting the following mammalianmethyltransferases: norepinephrine N-methyltransferase, histamineN-methyltransferase and catechol O-methyltransferase (see Fuller andNagarajan (1978), . Biochemical Pharmacology, 27:1981). Sinefungin isalso effective in inhibiting the S-adenosyl-methionine-dependentO-methyltransferase in avermectin-producing strains of Streptomycesavermitilis (see Schulman, et al. (1985), J. Antibiotics, 38:1494). Morerecently, sinefungin was reported effective in inhibiting anS-adenosylmethionine-dependent O-methyltransferase (macrocinO-methyltransferase) in Streptomyces fradiae (see Kreuzman, et al.(1988), J. Biological Chemistry, 263:15626). A method of usingsinefungin to inhibit an O-methyltransferase in strains of S. spinosa isdisclosed herein.

SUMMARY OF THE INVENTION

The present invention is directed to a new genus of the A83543 family ofcompounds, said genus including compounds of Formula 1 ##STR4## whereinR⁷ is hydrogen or a group of formula ##STR5## R⁸, R⁹, R¹⁰, R¹¹, and R¹²are independently hydrogen or methyl, provided that R¹¹ and R¹² are notconcurrently hydrogen; or an acid addition salt thereof when R⁷ is otherthan hydrogen.

In particular, this invention relates to new components of fermentationproduct A83543. The new components, termed Formula 2 compounds, have thefollowing general formula: ##STR6## wherein R¹³ is a group of formula##STR7## and R¹⁴, R¹⁵, R¹⁶, R¹⁷ and R¹⁸ are independently hydrogen ormethyl, provided that R¹⁷ and R¹⁸ are not concurrently hydrogen; or anacid addition salt thereof when R¹ is other than hydrogen.

Preferably, this invention relates to new A83543 components, Formula 2components, designated A83543K, A835430, A83543P, A83543U, A83543V,A83543W and A83543Y, wherein R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷ and R¹⁸ are foreach component as follows:

    ______________________________________                                        Component                                                                              R.sup.13                                                                              R.sup.14                                                                              R.sup.15                                                                            R.sup.16                                                                            R.sup.17                                                                            R.sup.18                           ______________________________________                                        K        (a)     CH.sub.3                                                                              H     CH.sub.3                                                                            CH.sub.3                                                                            CH.sub.3                           O        (a)     CH.sub.3                                                                              CH.sub.3                                                                            CH.sub.3                                                                            CH.sub.3                                                                            CH.sub.3                           F        (a)     CH.sub.3                                                                              H     CH.sub.3                                                                            CH.sub.3                                                                            H                                  U        (a)     CH.sub.3                                                                              H     CH.sub.3                                                                            H     CH.sub.3                           V        (a)     CH.sub.3                                                                              CH.sub.3                                                                            CH.sub.3                                                                            H     CH.sub.3                           W        (a)     CH.sub.3                                                                              CH.sub.3                                                                            CH.sub.3                                                                            CH.sub.3                                                                            H                                  Y        (a)     CH.sub.3                                                                              H     H     CH.sub.3                                                                            CH.sub.3                           ______________________________________                                    

Another aspect of this invention is a process for producing a compoundof Formula 1, which comprises culturing a strain of S. spinosa, selectedfrom strains NRRL 18395 (A83543.1), NRRL 18537 (A83543.3), NRRL 18538(A83543.4), NRRL 18539 (A83543.5), NRRL 18719 (A83543.6) and NRRL 18823(A83543.9) or a Formula 1-producing mutant thereof, in a suitableculture medium, containing from about 50 μg/ml to about 200 μg/ml ofsinefungin, under submerged aerobic conditions until a recoverableamount of a compound of Formula 1 is produced. The Formula 1 compound isextracted from the fermentation broth and from the mycelium with polarorganic solvents. The compound may be further purified by techniqueswell known in the art, such as column chromatography.

A still further aspect of the present invention is a process forproducing a compound of Formula 2 which comprises cultivating S. spinosastrain NRRL 18743 (A83543.8) or an A83543K-producing mutant thereof, Ina suitable culture medium, under submerged aerobic fermentationconditions, until a recoverable amount of a compound of Formula 1 isproduced. The Formula 1 compound can be isolated and purified asdescribed herein.

Because strain NRRL 18743 is a newly discovered strain, this inventionfurther provides a biologically purified culture of this microorganism.

The Formula 1 compounds are useful for the control of mites and insects,particularly Lepidoptera, Homoptera, and Diptera species. Therefore,insecticidal and miticidal compositions and methods for reducing thepopulations of insects and mites using these compounds are also a partof this invention.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the infrared absorption spectrum of A83543K in KBr.

FIG. 2 shows the proton nuclear magnetic resonance spectrum of A83543Kin acetone-d₆.

FIG. 3 shows the UV spectrum spectrum of A83543K in EtOH.

FIG. 4 shows the infrared absorption spectrum of A83543O in KBr.

FIG. 5 shows the proton nuclear magnetic resonance spectrum of A83543Oin acetone-d₆.

FIG. 6 shows the UV spectrum spectrum of A83543O in EtOH.

FIG. 7 shows the infrared absorption spectrum of A93543P in KBr.

FIG. 8 shows the proton nuclear magnetic resonance spectrum of A83543Pin acetone-d₆.

FIG. 9 shows the UV spectrum spectrum of A83543P in EtOH.

FIG. 10 show the infrared absorption spectrum of A83543U in KBr.

FIG 11 shows the proton nuclear magnetic resonance spectrum of A83543Uin acetone-d₆.

FIG. 12 shows the UV spectrum spectrum of A83543Q in EtOH.

FIG. 13 shows the infrared absorption spectrum of A83543V in KBr.

FIG. 14 shows the proton nuclear magnetic resonance spectrum of A83543Vin acetone-d₆.

FIG. 15 shows the UV spectrum spectrum of A83543V in EtOH.

FIG. 16 shows the infrared absorption spectrum of A83543W in KBr.

FIG. 17 shows the proton nuclear magnetic resonance spectrum of A83543Win acetone-d₆.

FIG. 18 shows the UV spectrum spectrum of A83543W in EtOH.

FIG. 19 shows the infrared absorption spectrum of A83543Y in KBr.

FIG. 20 shows the proton nuclear magnetic resonance spectrum of A83543Yin acetone-d₆.

FIG. 21 shows the UV spectrum spectrum of A83543Y in EtOH.

FIG. 22 shows the principle component plot of fatty acid analysis forstrains A83543.1, A83543.3, A83543.4, A83543.5, A83543.6, A83543.7,A83543.8 and A83543.9.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to compounds of the family of relatedcomponents produced by S. spinosa. The general structure of thecompounds of the present invention is shown in the following formula:##STR8## wherein R⁷ is hydrogen or a group of formula ##STR9## R⁸, R⁹,R¹⁰, R¹² are independently hydrogen or methyl, provided that R¹¹ and R¹²are not concurrently hydrogen; or an acid addition salt thereof when R⁷is other than hydrogen.

A preferred aspect of the invention is the Formula 1 compounds whereinR⁸ and R¹⁰ are methyl. A more preferred aspect of the invention is theFormula 1 compounds wherein R⁸ and R¹⁰ are methyl and R⁷ is a group offormula ##STR10##

Another aspect of the present invention is new components offermentation product A83543. These new A83543 components, termed Formula2 compounds, have the following chemical structure: ##STR11## whereinR¹³ is a group of formula ##STR12## and R¹⁴, R¹⁵, R¹⁶, R¹⁷ and R¹⁸ areindependently hydrogen or methyl, provided that R¹⁷ and R¹⁸ are notconcurrently hydrogen; or an acid addition salt thereof when R¹³ isother than hydrogen.

A more preferred aspect of the present invention is the Formula 2compounds wherein R¹⁴ is CH₃ and R¹³ is a group of formula ##STR13##

Preferably, this invention relates to new A83543 components, Formula 2compounds, designated A83543K, A83543O, A83543P, A83543U, A83543V,A83543W, and A83543Y, wherein R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷ and R¹⁸ areindividually for each new component as follows:

    ______________________________________                                        Component                                                                              R.sup.13                                                                              R.sup.14                                                                              R.sup.15                                                                            R.sup.16                                                                            R.sup.17                                                                            R.sup.18                           ______________________________________                                        K        (a)     CH.sub.3                                                                              H     CH.sub.3                                                                            CH.sub.3                                                                            CH.sub.3                           O        (a)     CH.sub.3                                                                              CH.sub.3                                                                            CH.sub.3                                                                            CH.sub.3                                                                            CH.sub.3                           P        (a)     CH.sub.3                                                                              H     CH.sub.3                                                                            CH.sub.3                                                                            H                                  U        (a)     CH.sub.3                                                                              H     CH.sub.3                                                                            H     CH.sub.3                           V        (a)     CH.sub.3                                                                              CH.sub.3                                                                            CH.sub.3                                                                            H     CH.sub.3                           W        (a)     CH.sub.3                                                                              CH.sub.3                                                                            CH.sub.3                                                                            CH.sub.3                                                                            H                                  Y        (a)     CH.sub.3                                                                              H     H     CH.sub.3                                                                            CH.sub.3                           ______________________________________                                    

The chemical structures of these new components were determined byspectrometric methods, including infrared spectroscopy (IR), nuclearmagnetic resonance spectroscopy (NMR), and ultraviolet spectroscopy(UV), and by comparison to the A83543 components (see Kirst, et al.(1991), supra). The following paragraphs describe the physical andspectral properties of components A83543K, A83543O, A83543P, A83543U,A83543V, A83543W and A83543Y.

For the convenience of the reader, the following diagram of A83543Kprovides the position designations of all NMR spectral data for theA83543 natural factors presented below: ##STR14## A83543K:

A83543K has the following characteristics:

Molecular weight: 717

Empirical formula: C₄₀ H₆₃ NO₁₀

UV (EtOH): 243 nm (ε=10,657)

MS (FAB): (M+H) m/z 718

IR (KBR): see FIG. 1.

Table I summarizes the ¹ H and ¹³ C NMR spectral data for A83453K (inacetone-d₆) as shown in FIG. 2. 8 37.24 1.97/1.38

                  TABLE I                                                         ______________________________________                                        .sup.1 H and .sup.13 C NMR data of A83543K in acetone-d.sub.6                 Position        .sup.13 C                                                                             .sup.1 H*                                             ______________________________________                                         1              172.69  --                                                     2              34.57   3.07/2.46                                              3              48.46   2.94                                                   4              42.41   3.50                                                   5              129.84  5.86                                                   6              130.39  5.92                                                   7              42.18   2.16                                                   8              37.24   1.97/1.38                                              9              77.09   4.35                                                  10              38.38   2.37/1.38                                             11              47.15   0.93                                                  12              50.49   2.85                                                  13              148.32  7.06                                                  14              145.78  --                                                    15              203.15  --                                                    16              48.41   3.31                                                  17              81.23   3.53                                                  18              35.18   1.50                                                  19              22.44   1.78/1.17                                             20              31.12   1.50                                                  21              76.86   4.65                                                  22              29.15   1.48                                                  23              9.56    0.81                                                  24              16.42   1.12                                                  1'              97.47   4.85                                                  2'              78.06   3.55                                                  3'              82.42   3.33                                                  4'              72.78   3.41                                                  5'              69.80   3.53                                                  6'              18.26   1.19                                                  2'-OCH.sub.3    59.02   3.42                                                  3'-OCH.sub.3    57.39   3.39                                                  1"              104.20  4.46                                                  2"              32.02   1.94/1.38                                             3"              18.93   1.81/1.48                                             4"              66.10   2.11                                                  5"              74.17   2.56                                                  6"              19.44   1.20                                                  N(CH.sub.3).sub.2                                                                             41.02   2.21                                                  ______________________________________                                         *Some assignments are from .sup.1 H/.sup.13 C correlations.              

A835430 has the following characteristics:

Molecular weight: 731

Empirical formula: C₄₁ H₆₅ NO₁₀

UV (EtOH): 243 nm (ε=9,267)

FD (M+) m/z 731

IR (KBr): see FIG. 3.

Table II summarizes the ¹ H and ¹³ C nuclear magnetic resonance (NMR)spectral data for A834540 (in acetone-d₆) as shown in FIG. 4.

                  TABLE II                                                        ______________________________________                                        .sup.1 H and .sup.13 C NMR data of A83543O in acetone-d.sub.6                 Position        .sup.13 C                                                                             .sup.1 H*                                             ______________________________________                                         1              172.60  --                                                     2              34.29   3.08/2.42                                              3              48.88   2.91                                                   4              42.71   3.45                                                   5              123.26  5.55                                                   6              137.16  --                                                     7              45.32   2.19                                                   8              35.52   2.02/1.45                                              9              76.80   4.64                                                  10              38.59   2.37/1.41                                             11              46.92   1.03                                                  12              49.94   2.78                                                  13              148.46  7.03                                                  14              145.07  --                                                    15              203.09  --                                                    16              48.39   3.30                                                  17              80.88   3.55                                                  18              35.00   1.50                                                  19              22.49   1.80/1.17                                             20              30.84   1.50                                                  21              76.50   4.34                                                  22              29.08   1.48                                                  23              9.54    0.80                                                  24              16.26   1.13                                                  6-CH.sub.3      20.85   1.73                                                  1'              97.21   4.87                                                  2'              77.80   3.56                                                  3'              82.23   3.33                                                  4'              72.54   3.41                                                  5'              69.61   3.55                                                  6'              18.21   1.19                                                  2'-OCH.sub.3    58.96   3.41                                                  3'-OCH.sub.3    57.31   3.39                                                  1"              104.02  4.46                                                  2"              31.85   1.94/1.39                                             3"              18.74   1.82/1.52                                             4"              65.90   2.12                                                  5"              73.90   3.57                                                  6"              19.39   1.20                                                  N(CH.sub.3).sub.2                                                                             40.92   2.20                                                  ______________________________________                                         *Values were taken from a heteronuclear one bond 2D correlation spectrum.

A83543P has the following characteristics:

Molecular weight: 703

Empirical formula: C₃₉ H₆₁ NO₁₀

UV (EtOH): 243 nm (ε=13,760)

MS (FAB): (M+H) m/z 704

IR (KBr): see FIG. 5.

Table III summarizes the ¹ H and ¹³ C nuclear magnetic resonance (NMR)spectral data for A83454P (in acetone-d₆) as shown in FIG. 6.

                  TABLE III                                                       ______________________________________                                        .sup.1 H and .sup.13 C NMR data of A83543P in acetone-d.sub.6                 Position        .sup.13 C                                                                             .sup.1 H*                                             ______________________________________                                         1              172.62  --                                                     2              34.43   3.06/2.44                                              3              48.74   2.94                                                   4              42.26   3.50                                                   5              129.70  5.86                                                   6              130.30  5.89                                                   7              42.06   2.14                                                   8              37.15   1.97/1.34                                              9              76.84   4.34                                                  10              38.28   2.36/1.36                                             11              47.05   0.92                                                  12              50.37   2.86                                                  13              148.43  7.03                                                  14              144.85  --                                                    15              203.09  --                                                    16              48.35   3.31                                                  17              80.96   3.55                                                  18              35.06   1.50                                                  19              22.44   1.78/1.16                                             20              30.91   1.55                                                  21              76.84   4.64                                                  22              29.11   1.47                                                  23              9.55    0.80                                                  24              16.29   1.11                                                  1'              96.68   4.86                                                  2'              82.07   3.33                                                  3'              72.41   3.63                                                  4'              74.27   3.30                                                  5'              69.45   3.53                                                  6'              18.17   1.19                                                  2'-OCH.sub.3    59.12   3.41                                                  1"              104.08  4.45                                                  2"              31.89   1.92/1.37                                             3"              18.72   1.81/1.52                                             4'              65.97   2.11                                                  5"              74.03   3.56                                                  6"              19.39   1.19                                                  N(CH.sub.3).sub.2                                                                             40.95   2.20                                                  ______________________________________                                         *Values were taken from a heteronuclear one bond 2D correlation spectrum.

A83543U:

A83543U has the following characteristics:

Molecular weight: 703

Empirical formula: C₃₉ H₆₁ NO₁₀

UV (EtOH): 242 nm (ε=17,095)

MS (FAB): (M+H) m/z 704

IR (KBr): see FIG. 7

Table IV summarizes the ¹ H and ¹³ C nuclear magnetic resonance (NMR)spectral data for A83454U (in acetone-d₆) as shown in FIG. 8.

                  TABLE IV                                                        ______________________________________                                        .sup.1 H and .sup.13 C NMR data of A83543U in acetone-d.sub.6                 Position        .sup.13 C                                                                             .sup.1 H*                                             ______________________________________                                         1              172.68  --                                                     2              34.53   3.10/2.49                                              3              48.41   2.97                                                   4              42.15   3.54                                                   5              129.77  5.91                                                   6              130.39  5.93                                                   7              42.37   2.19                                                   8              37.16   2.00/1.41                                              9              76.81** 4.38                                                  10              38.82   2.41/1.42                                             11              47.13   0.97                                                  12              50.46   2.91                                                  13              148.37  7.09                                                  14              144.96  --                                                    15              203.10  --                                                    16              48.63   3.35                                                  17              81.15   3.57                                                  18              35.15   1.57/1.51                                             19              22.45   1.82/1.21                                             20              31.06   1.58/1.49                                             21              76.85** 4.69                                                  22              29.04   1.54/1.50                                             23              9.57    0.83                                                  24              16.39   1.16                                                  1'              99.92   4.80                                                  2'              68.34   3.97                                                  3'              82.36   3.29                                                  4'              72.45   3.48                                                  5'              69.42   3.62                                                  6'              18.21   1.24                                                  3'-OCH.sub.3    57.06   3.42                                                  1"              104.16  4.49                                                  2"              31.98   1.97/1.42                                             3"              18.67   1.86/1.55                                             4"              66.06   2.14                                                  5"              74.13   3.60                                                  6"              19.43   1.24                                                  N(CH.sub.3).sub.2                                                                             41.01   2.24                                                  ______________________________________                                         *Values were taken from 1D or inverse 2D one bond correlation spectrum.       **Assignments may be reversed.                                           

A83543V:

A83543V has the following characteristics:

Molecular weight: 717

Empirical formula: C₄₀ H₆₃ NO₁₀

UV (EtOH): 242 nm (ε=10,140)

MS (FAB): (M+H) m/z 718

IR (KBr): see FIG. 9.

Table V summarizes the ¹ H and ¹³ C nuclear magnetic resonance (NMR)spectral data for A83454V (in acetone-d₆) as shown in FIG. 10.

                  TABLE V                                                         ______________________________________                                        .sup.1 H and .sup.13 C NMR data of A83543V                                    in acetone-d.sub.6                                                            Position       .sup.13 C*                                                                            .sup.l H*                                              ______________________________________                                         1             172.63  --                                                      2             34.34   3.10/2.45                                               3             48.88   2.88                                                    4             42.63   3.49                                                    5             123.20  5.57                                                    6             137.25  --                                                     6-CH.sub.3     20.77   1.76                                                    7             45.23   2.21                                                    8             35.44   2.02/1.45                                               9             76.22   4.36                                                   10             38.63   2.40/1.41                                              11             46.89   1.07                                                   12             49.92   2.80                                                   13             148.54  7.08                                                   14             145.10  --                                                     15             203.11  --                                                     16             48.46   3.34                                                   17             80.80   3.56                                                   18             35.12   1.54/1.50                                              19             22.50   1.82/1.21                                              20             30.81   1.56/1.51                                              21             76.71   4.67                                                   22             29.01   1.51                                                   23             9.38    0.82                                                   24             16.17   1.14                                                   1'             99.88   4.67                                                   2'             69.03   3.69                                                   3'             82.19   3.28                                                   4'             72.21   3.46                                                   5'             68.18   3.61                                                   6'             18.05   1.22                                                   3'-OCH.sub.3   56.98   3.41                                                   1"             104.14  4.49                                                   2"             31.91   1.95/1.41                                              3"             18.62   1.84/1.54                                              4"             65.92   2.14                                                   5"             73.92   3.59                                                   6"             19.31   1.22                                                   N(CH.sub.3).sub.2                                                                            40.78   2.23                                                   ______________________________________                                         *Values were taken from 1D and 2D inverse experiments                    

A83543W:

A83543W has the following characteristics:

Molecular weight: 717

Empirical formula: C₄₀ H₆₃ NO₁₀

UV (EtOH): 244 nm (ε=10,254)

MS (FAB): (M+H) m/z 718

IR (KBr): see FIG. 11

Table VI summarizes the ¹ H and ¹³ C nuclear magnetic resonance (NMR)spectral data for A83543W (in acetone-d₆) as shown in FIG. 12.

                  TABLE VI                                                        ______________________________________                                        .sup.1 H and .sup.13 C NMR data of                                            A83543W in acetone-d.sub.6                                                    Position       .sup.13 C*                                                                            .sup.l H*                                              ______________________________________                                         1             172.62  --                                                      2             34.46   3.08/2.44                                               3             49.00   2.90                                                    4             42.69   3.46                                                    5             123.52  5.55                                                    6             137.25  --                                                     6-CH.sub.3     20.93   1.76                                                    7             45.58   2.21                                                    8             35.83   2.04/1.46                                               9             76.67   4.35                                                   10             38.81   2.39/1.41                                              11             47.27   1.04                                                   12             50.09   2.80                                                   13             146.41  7.04                                                   14             145.15  --                                                     15             203.11  --                                                     16             48.45   3.32                                                   17             81.05   3.56                                                   18             35.19   1.51                                                   19             22.62   1.81/1.19                                              20             31.08   1.51                                                   21             76.94   4.66                                                   22             29.31   1.49                                                   23             9.58    0.80                                                   24             16.26   1.11                                                   1'             96.94   4.88                                                   2'             82.42   3.34                                                   3'             72.56   3.64                                                   4'             74.48   3.32                                                   5'             69.54   3.56                                                   6'             18.27   1.20                                                   2'-OCH.sub.3   59.13   3.43                                                   1"             104.34  4.47                                                   2"             32.13   1.96/1.40                                              3"             18.84   1.83/1.54                                              4"             66.26   2.12                                                   5"             74.20   3.59                                                   6"             19.56   1.21                                                   N(CH.sub.3).sub.2                                                                            41.15   2.22                                                   ______________________________________                                         *Values were taken from .sup.1 H/.sup.13 C inverse one bond correlation       spectra.                                                                 

A83543Y has the following characteristics:

Molecular weight: 703

Empirical formula: C₃₉ H₆₁ NO₁₀

UV (EtOH): 243 nm (ε=14,042)

MS (FAB): (M+H) m/z 704

IR (KBr): see FIG. 11

Table VII summarizes the ¹ H and 13C nuclear magnetic resonance (NMR)spectral data for A83543Y (in acetone-d₆) as shown in FIG. 12.

                  TABLE VII                                                       ______________________________________                                        .sup.1 H and .sup.13 C NMR data of                                            A83543Y in acetone-d.sub.6                                                    Position       .sup.13 C*                                                                            .sup.1 H*                                              ______________________________________                                         1             172.42  --                                                      2             34.86   3.07/2.42                                               3             48.80   2.96                                                    4             42.04** 3.44                                                    5             129.68  5.87                                                    6             130.32  5.91                                                    7             42.00** 2.16                                                    8             37.08   1.98/1.38                                               9             76.86   4.35                                                   10             38.26   2.38/1/39                                              11             47.07   0.94                                                   12             50.30   2.87                                                   13             148.45  7.06                                                   14             144.72  --                                                     15             203.06  --                                                     16             47.97   3.35                                                   17             81.23   3.56                                                   18             34.86   1.61/1.52                                              19             22.22   1.78/1.19                                              20             33.56   1.54/1.47                                              21             72.97   4.69                                                   22             21.58   1.12                                                   23             --      --                                                     24             16.42   1.13                                                   1'             97.24   4.85                                                   2'             77.81   3.55                                                   3'             82.25   3.31                                                   4'             72.61   3.41                                                   5'             69.64   3.55                                                   6'             18.21   1.19                                                   2'-OCH.sub.3   58.94   3.41                                                   3'-OCH.sub.3   57.28   3.40                                                   1"             104.16  4.47                                                   2"             31.90   1.94/1.41                                              3"             18.71   1.82/1.53                                              4"             65.94   2.12                                                   5"             74.02   3.57                                                   6"             19.37   1.21                                                   N(CH.sub.3).sub.2                                                                            40.93   2.22                                                   ______________________________________                                         *Data obtained from 1D, inverse heteronuclear correlation, homonuclear        decoupling and COSY experiments.                                              **Assignments may be reversed.                                           

Components A83543K, A83543O, A83543P, A83543U, A83543V, A83543W andA83543Y are structurally distinct from previously described compounds.The present compounds possess neutral sugars which have not beenpreviously described: components A83543K, A83543O and A83543Y have aneutral sugar identified as α-2,3-di-O-methylrhamnose; componentsA83543P and A83543W have a neutral sugar identified as 2-O-methylrhamnose; components A83543U and A83543V have a neutral sugaridentified as 3-O-methylrhamnose.

The amino sugar can be selectively removed from the new A83543components to give new A83543 pseudoaglycones, termed Formula 3compounds. These compounds are a further aspect of the present inventionand are the compounds of Formula 1 wherein R¹ is hydrogen.

The selective removal of the amino sugar from A83543K, A83543O, A83543P,A83543U, A83543V, A83543W and A83543Y produces A83543K pseudoaglycone,A83543O pseudoaglycone, A83543P pseudoaglycone, A83543U pseudoaglycone,A83543V pseudoaglycone, A83543W pseudoaglycone, and A83543Ypseudoaglycone respectively. These compounds are shown in the followingformula: ##STR15##

    ______________________________________                                        Compound         R.sup.19                                                                             R.sup.20 R.sup.21                                                                           R.sup.22                                ______________________________________                                        A83543K pseudoaglycone                                                                         H      CH.sub.3 CH.sub.3                                                                           CH.sub.3                                A83543O pseudoaglycone                                                                         CH.sub.3                                                                             CH.sub.3 CH.sub.3                                                                           CH.sub.3                                A83543P pseudoaglycone                                                                         H      CH.sub.3 CH.sub.3                                                                           H                                       A83543U pseudoaglycone                                                                         H      CH.sub.3 H    CH.sub.3                                A83543V pseudoaglycone                                                                         CH.sub.3                                                                             CH.sub.3 H    CH.sub.3                                A83543W pseudoaglycone                                                                         CH.sub.3                                                                             CH.sub.3 CH.sub.3                                                                           H                                       A83543Y pseudoaglycone                                                                         H      H        CH.sub.3                                                                           CH.sub.3                                ______________________________________                                    

The Formula 2 compounds are used to prepare the Formula 3 compounds bythe reaction of a Formula 2 compound with acid to remove the aminosugar. Suitable acids include hydrochloric and sulfuric, the preferredacid for the transformation is sulfuric. The reaction is preferablycarried out in a polar organic solvent, a mixture of a polar organicsolvent and water, or water. Suitable organic solvents include methanol,THF, acetonitrile and dioxane. The preferred solvents for thetransformation are a mixture of methanol and water or water. Thereaction may be carried out at a temperature from about 25° C. to about95° C., preferably at 80° C.

The pseudoaglycones are useful as starting materials for the preparationof new A83543 compounds, for example, the pseudoaglycone may beglycosylated at the hydroxyl group where the amino sugar was present.This glycosylation may be carried out by chemical synthesis or bymicrobial bioconversion.

Another aspect of the present invention is the chemical demethylation ofcertain Formula 1 compounds. The Formula 1 compounds may be grouped into3 subgroups: 1A, 1B and 1C. The Formula 1A compounds are the Formula 1compounds wherein R⁷ is a group of formula: ##STR16##

The Formula 1B compounds are the Formula 1 compounds wherein R⁷ is agroup of formula: ##STR17##

The Formula 1C compounds are the Formula 1 compounds wherein R⁷ is agroup of formula: ##STR18##

As described herein, the Formula 1B compounds may be prepared from theFormula 1A compounds. Similarly, the Formula 1C compounds may beprepared from the Formula 1B compounds. These compounds may be preparedby chemical demethylation of a corresponding new A83543 component. Eachof these sub-groups is also a subset of the Formula 2 compounds.

The N-demethyl derivatives, the Formula 1B compounds, are prepared bythe reaction of a Formula 1A compound with iodine. The reaction iscarried out in a polar organic solvent, such as methanol, or a mixtureof a polar organic solvent and water, such as aqueous methanol. When thereaction is carried out in aqueous methanol, a buffer may be added tothe solvent mixture. A preferred buffer is sodium acetate. The reactionis preferably carried out at a temperature from about 30° C. about 70°C. for about 2 to about 6 hours.

The di-N-demethyl derivatives, the Formula 1C compounds, may be preparedby the reaction of a Formula 1A and Formula 1B compound with sodiummethoxide/iodine. The reaction is preferably carried out in a polarorganic solvent, such as methanol. Further, the reaction is carried outat a temperature from about 10° C. to about 15° C., preferably between0° C. to 5° C. The reaction times vary from about 4 hours to about 6hours.

Illustrative examples of the Formula 1B and 1C compounds are shown inthe following formula: ##STR19##

    ______________________________________                                        Compound      R.sup.7                                                                              R.sup.9 R.sup.10                                                                            R.sup.11                                                                            R.sup.12                             ______________________________________                                        N-demethyl-A83543K                                                                          (b)    H       CH.sub.3                                                                            CH.sub.3                                                                            CH.sub.3                             di-N-demethyl-A83543K                                                                       (c)    H       CH.sub.3                                                                            CH.sub.3                                                                            CH.sub.3                             N-demethyl-A83543O                                                                          (b)    CH.sub.3                                                                              CH.sub.3                                                                            CH.sub.3                                                                            CH.sub.3                             di-N-demethyl-A83543O                                                                       (c)    CH.sub.3                                                                              CH.sub.3                                                                            CH.sub.3                                                                            CH.sub.3                             N-demethyl-A83543P                                                                          (b)    H       CH.sub.3                                                                            CH.sub.3                                                                            H                                    di-N-demethyl-A83543P                                                                       (c)    H       CH.sub.3                                                                            CH.sub.3                                                                            H                                    N-demethyl-A83543U                                                                          (b)    H       CH.sub.3                                                                            H     CH.sub.3                             di-N-demethyl-A83543U                                                                       (c)    H       CH.sub.3                                                                            H     CH.sub.3                             N-demethyl-A83543V                                                                          (b)    CH.sub.3                                                                              CH.sub.3                                                                            H     CH.sub.3                             di-N-demethyl-A83543V                                                                       (c)    CH.sub.3                                                                              CH.sub.3                                                                            H     CH.sub.3                             N-demethyl-A83543W                                                                          (b)    CH.sub.3                                                                              CH.sub.3                                                                            CH.sub.3                                                                            H                                    di-N-demethyl-A83543W                                                                       (c)    CH.sub.3                                                                              CH.sub.3                                                                            CH.sub.3                                                                            H                                    N-demethyl-A83543Y                                                                          (b)    H       H     CH.sub.3                                                                            CH.sub.3                             di-N-demethyl-A83543Y                                                                       (c)    H       H     CH.sub.3                                                                            CH.sub.3                             ______________________________________                                    

The Formula 2 compounds, which are the Formula 1 compounds wherein R⁷ isother than hydrogen, can react to form various salts, which are also apart of this invention. These salts are useful, for example, inseparating and purifying the Formula 2 compounds. In addition, some ofthe salt forms may have increased water solubility. These salts areprepared using standard procedures for salt preparation. For example,A83543K can be neutralized with an appropriate acid to form an acidaddition salt.

The acid addition salts are particularly useful. Representative suitablesalts include those salts formed by standard reactions with both organicand inorganic acids such as, for example, sulfuric, hydrochloric,phosphoric, acetic, succinic, citric, lactic, maelic, fumaric, cholic,pamoic, mucic, glutamic, camphoric, glutaric, glycolic, phthalic,tartaric, formic, lauric, stearic, salicyclic, methanesulfonic,benzenesulfonic, sorbic, picric, benzoic, cinnamic, and like acids.

For convenience in the discussions which follow, A83543A-producingstrains have been given the following designations: A83543.1, A83543.3,A83543.4, and A83543.5. Also, a new A83543K-producing strain has beengiven the designation A83543.8. Cultures A83543.1, A83543.3, A83543.4,A83543.5, A83543.6, A83543.7, A83543.8 and A83543.9 have been depositedand made a part of the stock culture collection of the Midwest AreaRegional Research Center, Agricultural Research Service, United StatesDepartment of Agriculture, from which they are available to the publicunder the following accession numbers:

    ______________________________________                                               NRRL No.      Strain No.                                               ______________________________________                                               18395         A83543.1                                                        18537         A83543.3                                                        18538         A83543.4                                                        18539         A83543.5                                                        18719         A83543.6                                                        18720         A83543.7                                                        18743         A83543.8                                                        18823         A83543.9                                                 ______________________________________                                    

Culture A83543.1 was obtained by chemical mutation of culture A83543,which was isolated from a soil sample collected in the Virgin Islands.Mertz and Yao (1990), Int'l J. of Systematic Bacteriology, 40:34.Culture 83543.4 was derived from culture A83543.1. Each of the strainsA83543.3, A83543.4, A83543.5, A83543.6, and A83543.7 was derived fromA83543.1 by chemically-induced mutagenesis withN-methyl-N'-nitro-N-nitrosoguanidine. Strains A83543.8 and A83543.9 werederived from A83453.4 by chemically-induced mutagenesis withN-methyl-N'-nitro-N-nitrosoguanidine. Except for differences in theproduction of the A83543 components, these isolates appear the same asthe parent culture.

Cultural Characteristics

Cultures A83543.1, A83543.3, A83543.4, A83543.5, A83543.6, A83543.7,A83543.8 and A83543.9 were grown on twelve agar plating media andcompared for growth, reverse color, aerial hyphae production, spore masscolor, and soluble pigment production. No significant differences wereobserved on any of the media used. The cultures grew well on bothcomplex and defined media. Aerial hyphae were produced on most of themedia used. The aerial spore mass color was predominantly white, and thereverse side was yellow to yellow-brown. No distinctive pigmentation waspresent; however, a soluble brown pigment was released into some media.The cultural characteristics of A83543.3, A83543.4, A83543.5, A83543.6,A83543.7, A83543.8, and A83543.9 are similar to the original taxonomicdescription of A83543.1 (see Mertz and Yao (1990), supra).

Morphological Characteristics P Well-formed aerial hyphae, which weresegmented into long chains of spores arranged as hooks and open loops,were present on most of the media. Spirals were also observed, but theywere short and incomplete. The general morphology was rectus-flexibilis.Aerial hyphae of each of the strains had a distinctive bead-likeappearance, with many empty spaces in the spore chain. This featuredemonstrated that a spore sheath encased the spore chain, which is adistinctive feature of the genus Saccharopolyspora. Except fordifferences in the production of the A83543 components, these isolatesappear similar to the parent culture.

Physiological Characteristics P Fatty acid analyses from each of thestrains were compared. Cells were grown for 96 hours at 28° C. intrypticase soy broth (Difco Laboratories, Detroit, Mich.). Fatty acidmethyl esters were analyzed by gas-liquid chromatography with a model5898A computer-controlled gas-liquid chromatography system(Hewlett-Packard Co., Palo Alto, Calif.) (see Miller and Berger,"Bacterial Identification by Gas Chromatography of Whole Cell FattyAcids," Hewlett-Packard Application Note 228-41. These results arepresented in Table VIII).

                                      TABLE VIII                                  __________________________________________________________________________    Fatty Acid                                                                           A83543.1                                                                           A83543.3                                                                           A83543.4                                                                           A83543.5                                                                           A83543.6                                                                           A83543.7                                                                           A83543.8                                                                           A83543.9                            __________________________________________________________________________    15:0 ISO                                                                             15.95     22.47     16.49                                                                              17.00                                                                              19.76                                                                              17.42                               16:0 ISO                                                                             28.71     22.00     25.76                                                                              27.39                                                                              23.14                                                                              24.34                               16:1 Cis 9                                                                           --        1.35      --   --   0.90 0.92                                15:0 ISO 2OH                                                                         2.67      2.02      3.87 3.95 2.44 1.78                                16:0   1.20      0.69      0.63 0.60 0.47 0.36                                17:0 ISO F.sup.1                                                                     5.52      8.62      7.54 5.51 7.55 8.72                                17:0 Iso                                                                             13.55     20.67     16.40                                                                              13.89                                                                              21.15                                                                              19.43                               17:0 Anteiso                                                                         8.39      3.94      4.69 5.18 3.57 5.52                                17:1 B 4.14      3.97      4.65 6.68 4.47 4.61                                17:1 C 2.52      2.88      4.90 5.53 3.18 3.02                                17:0   4.26      1.49      3.13 3.84 2.25 1.67                                16:1 2OH                                                                             1.87      1.52      1.93 0.92 1.36 2.17                                18:1 Iso F                                                                           6.55      4.16      5.82 6.00 5.75 5.74                                18:1 Cis 9                                                                           0.34      1.03      0.64 0.63 0.96 0.84                                __________________________________________________________________________     .sup.1 F, B and C indicate double bond positions or configurations that       are unknown.                                                             

Principal-component analysis is a branch of multivariate statistics thatdeals with internal relationships of a set of variables. In thisanalysis, the greatest amount of variance within the original data ortest results is expressed as principal components (see Alderson, "TheApplication and Relevance of Nonheirarchic Methods in BacterialTaxonomy", in Computer-Assisted Bacterial Systematics 227 (1985)). Aplot showing scatter or variability can be constructed. Relationshipscan be evaluated by examining the variance, and a microbial populationcharacterized. A two-dimensional principal component plot from the fattyacid analyses of strains A83543.1, A83543.3, A83543.4, A83543.5,A83543.6, A83543.7, A83543.8 and A83543.9 is shown in FIG. 13. Thevalues refer to the degrees of separation between the strains involved.The differences between the strains are not taxonomically significant.

As is the case with other organisms, the characteristics of theA83543-producing strains are subject to variation. Thus, mutants ofthese strains may be obtained by physical and chemical methods known inthe art. For example, other strains may be obtained by treatment withchemicals such as N-methyl-N'-nitro-N-nitrosoguanidine. Natural andinduced mutants of the S. spinosa NRRL 18395, NRRL 18537, NRRL 18538,NRRL 18539 NRRL 18719, NRRL 18720, NRRL 18743 and NRRL 18823 strains,which retain the characteristic of producing recoverable amounts of aFormula 1 compound, when cultured in appropriate conditions areapplicable in the present invention.

One aspect of the present invention is the production of a compound ofFormula 1 produced by culturing an A83543A-producing strain of S.spinosa in a suitable culture medium containing sinefungin, selectedfrom the group consisting of NRRL 18395, NRRL 18537, NRRL 18538, andNRRL 18539 or an A83543A-producing mutant thereof. An "A83543A-producingmutant" is a strain derived from any one of the A83543A-producingstrains of S. spinosa, NRRL 18395, NRRL 18537, NRRL 18538, NRRL 18539,which is capable of producing recoverable amounts of A83543A and whichis capable, when cultured in a suitable culture medium containingsinefungin, to produce concomitant amounts of A83543K and A83543O.

Another aspect of the present invention is the production of a compoundof Formula 1 by culturing an A83543H-producing strain of S. spinosa,such as NRRL 18823 or an A83543H-producing mutant thereof, in a suitableculture medium containing sinefungin. An "A83543H-producing mutant" is astrain derived from any one of the A83543H-producing strains of S.spinosa, NRRL 18823, which is capable of producing recoverable amountsof A83543H and which is capable, when cultured in a suitable culturemedium containing sinefungin, to produce concomitant amounts of A83543Uand A83543V.

A still further aspect of this invention is the production of a compoundof Formula 1 by culturing an A83543J-producing strain of S. spinosa,such as NRRL 18719 or an A83543J-producing mutant thereof, in a suitableculture medium containing sinefungin. An "A83543J-producing mutant" is astrain derived from any one of the A83543J-producing strains of S.spinosa, NRRL 18719 or NRRL 18720, which is capable of producingrecoverable amounts of A83543J and which is capable, when cultured in asuitable culture medium containing sinefungin, to produce concomitantamounts of A83543P and A83543W.

Typically, sinefungin is added to the production medium after 48-72hours or for large scale production, the addition of sinefungin ispostponed until the culture begins to grow as indicated by the uptake ofoxygen. Preferably, sinefungin is added to the fermentation medium about48 hours to about 72 hours after inoculation. Sinefungin may be added asa solid or as a solution. For convenience, when sinefungin is added to alarge scale fermentation, addition as an alcoholic solution ispreferred. Such a solution is prepared by dissolving sinefungin in asufficient volume of methyl alcohol, then sterilizing the solution byfiltration through a 0.45 μfilter.

Alternatively, the Formula 1 compounds are produced by culturing S.spinosa strain NRRL 18743 (which produces components A83543K, A83543Oand A83543Y), or an A83543K-producing mutant thereof, in a suitableculture medium without the addition of sinefungin. An "A83543K-producingmutant" is a strain derived from S. spinosa NRRL 18743 which is capableof producing recoverable amounts of A83543K.

After production, the Formula 1 compound may be separated from theculture medium using various isolation and purification procedures whichare well understood in the art. For economy in production, optimalyield, and ease of product isolation, certain culture media arepreferred. For example, preferred carbon sources in large-scalefermentation are glucose and methyl oleate, although ribose, xylose,fructose, galactose, mannose, mannitol, soluble starch, potato dextrin,oils such as soybean oil and the like can also be used. Preferrednitrogen sources are cottonseed flour, peptonized milk and corn steepliquor, although fish meal, digested soybean meal, yeast extract,enzyme-hydrolyzed casein, beef extract, and the like can also be used.Among the nutrient inorganic salts which can be incorporated in theculture media are the customary soluble salts capable of yielding zinc,sodium, magnesium, calcium, ammonium, chloride, carbonate, sulfate,nitrate and like ions. Essential trace elements necessary for the growthand development of the organism should also be included in the culturemedium. Such trace elements commonly occur as impurities in othersubstituents of the medium in amounts sufficient to meet the growthrequirements of the organism.

Usually, if foaming is a problem, small amounts (i.e., 0.2 ml/L) of anantifoam agent such as polypropylene glycol may be added to large-scalefermentation media. In the case of the A83543-producing cultures,however, conventional defoamers inhibit A83543 production. Foaming canbe controlled by including soybean oil or PLURONIC L-101 (BASF,Parsipanny, N.J.) in the medium (1-3%). Additional oil may be added iffoaming develops.

For production of substantial quantities of a Formula 1 compound,submerged aerobic fermentation in stirred bioreactors is preferred;however, small quantities of a Formula 1 compound may be obtained byshake-flask culture. Because of the time lag in production commonlyassociated with inoculation of large bioreactors with the spore form ofthe organism, it is preferable to use a vegetative inoculum. Thevegetative inoculum is prepared by inoculating a small volume of culturemedium from a stock culture preserved in liquid nitrogen to obtain afresh, actively growing culture of the organism. The vegetative inoculumis then transferred to a larger bioreactor. The vegetative inoculummedium can be the same as that used for larger fermentations, but othermedia are also suitable.

The Formula 1 compound is produced by the A83543-producing strains whengrown at temperatures between about 24° C. and about 33° C. Optimumtemperatures for production appear to be about 28°-30° C.

As is customary in submerged aerobic culture processes, sterile air isblown into the vessel from the bottom while the medium is stirred withconventional turbine impellers. In general, the aeration rate andagitation rate should be sufficient to maintain the level of dissolvedoxygen at or above 80%, with an internal vessel pressure of about 0.34atmospheres.

Production of the Formula 1 compound can be followed during thefermentation by testing extracts of the broth. A preferred method forfollowing the production is analysis of the broth extracts by highperformance liquid chromatography (HPLC). A suitable system for analysisis described in Example 1.

Following the production in shake flasks or in stirred reactors, theFormula 1 compound can be recovered from the fermentation medium bymethods used in the art. The compounds produced during fermentation ofthe A83543-producing strain occur in both the mycelia and the broth. TheFormula 1 compounds are lipophilic; when a substantial amount of oil isused in the fermentation, whole broth extraction is more efficient. Ifonly small amounts of oil are used, the major portion of the Formula 1compound is present in the mycelia. In that case, more efficientrecovery of the Formula 1 compound is accomplished by initiallyfiltering the medium to separate the broth from the mycelial mass (thebiomass).

The Formula 1 compound can be recovered from the biomass by a variety oftechniques. A suitable technique involves washing the separated biomasswith water to remove remaining broth, mixing the biomass with a polarsolvent in which the Formula 1 compound is soluble, e.g., methanol oracetone, separating and concentrating the solvent, extracting theconcentrate with a non-polar solvent and/or adsorbing it onto areverse-phase silica gel adsorbent, such as reverse phase C₈ or C₁₈resin, or a high porous polymer such as HP-20 or HP-20ss (MitsubishiChemical Industries Co., Ltd., Japan). The active material is elutedfrom the adsorbent with a suitable solvent such as, for example, H₂₀O:acetonitrile:methanol mixtures, optionally containing small amounts ofTHF.

A preferred technique for isolating the Formula 1 compound from thebiomass involves adding an equal volume of acetone to the whole broth,filtering the mixture in a ceramic filter to remove the biomass, andextracting the filtrate with ethyl acetate. The ethyl acetate extract isconcentrated in vacuo to remove the acetone, and the aqueous layer isseparated from the organic layer. The ethyl acetate solution is furtherconcentrated in vacuo, and the concentrate is extracted with diluteaqueous acid (pH 3). The Formula 1 compound may be further purified bychromatography as described herein.

A more preferred technique for isolating the Formula 1 compound from thebiomass involves adding an equal volume of acetone to the whole broth,filtering the mixture in a ceramic filter to remove the biomass, andadjusting the pH of the filtrate to about pH 9 to about pH 13. Thissolution is applied to HP-20ss (Mitsubishi Chemical Industries Co.,Ltd., Japan) and the column washed with a mixture of methanol,acetonitrile, and water (1:1:2). The Formula 1 compound is eluted with a95:5 mixture of methanol/acetonitrile (1:1) containing 0.1% ammoniumacetate (pH 8.1). The fractions containing the Formula 1 compounds arecombined and lyophilized. The Formula 1 compound may be further purifiedby chromatography as described herein.

Alternatively, the culture solids, including medium constituents andmycelium, can be used without extraction or separation, but preferablyafter removal of water, as a source of the Formula 1 compound. Forexample, after production of the Formula 1 compound, the wholefermentation broth can be dried by lyophilization, by drum-drying, or byazeotropic distillation and drying. The dried broth can then be useddirectly, for example, by mixing it directly into feed premix or intoformulations for sprays and powders.

Insecticide and Miticide Activity The Formula 2 compounds are useful forthe control of insects and mites. Therefore, a further aspect of thepresent invention is directed to methods for inhibiting an insect ormite which comprises applying to the locus of the mite or insect aninsect- or mite-inhibiting amount of a Formula 2 compound.

The "locus" of the insect or mite refers to the environment in which theinsect or mite lives or where its eggs are present, including the airsurrounding it, the food it eats, or objects which it contacts. Forexample, plant-ingesting insects or mites can be controlled by applyingthe active compound to plant parts which the insects or mites eat orinhabit, particularly the foliage.

The term "inhibiting an insect or mite" refers to a decrease in thenumber of living insects or mites or to a decrease in the number ofviable insect or mite eggs. The extent of reduction accomplished by acompound depends, of course, upon the application rate of the compound,the particular compound used, and the target insect or mite species. Atleast an insect-inactivating or mite-inactivating amount should be used.

The terms "insect-inactivating amount" and "mite-inactivating amount"are used to describe the amount which is sufficient to cause ameasurable reduction in the treated insect or mite population.Generally, an amount in the range from about 1 to about 1,000 ppm (or0.01 to 1 kg/a) of active compound is used.

The Formula 2 compounds show activity against a number of insects andmites. More specifically, the compounds show activity against beetarmyworm and tobacco budworm, which are members of the insect orderLepidoptera. Other typical members of this order are southern armyworm,codling moth, cutworms, clothes moths, Indian meal moth, leaf rollers,corn ear worm, cotton bollworm, European corn borer, imported cabbageworm, cabbage looper, pink bollworm, bagworms, Eastern tent caterpillar,sod webworm, and fall armyworm.

The Formula 2 compounds also show activity against leaf hoppers, whichis a member of the insect order Homoptera. Other members of this orderinclude cotton aphid, plant hoppers, pear psylla, apple sucker, scaleinsects, whiteflies, and spittle bugs, as well as a number of otherhost-specific aphid species.

In addition, the Formula 2 compounds show activity against stable flies,blowflies, and mosquitoes, which are members of the insect orderDiptera. Another typical member of this order is the common house fly.

The Formula 2 compounds also show activity against two-spotted spidermites, which is a member of the insect order Acarina. Other typicalmembers of this order include mange mite, scab mite, sheep scab mite,chicken mite, scalyleg mite, depluming mite, and dog follicle mite.

The Formula 2 compounds are useful for reducing populations of insectsand mites and are used in a method of inhibiting an insect or mitepopulation which comprises applying to a locus of the insect or mite aneffective insect- or mite-inactivating amount of a Formula 2 compound.In one preferred embodiment, the present invention is directed to amethod for inhibiting a susceptible insect of the order Lepidopterawhich comprises applying to a plant an effective insect-inactivatingamount of a Formula 2 compound in accordance with the present invention.Another preferred embodiment of the invention is directed to a method ofinhibiting biting flies of the order Diptera in animals which comprisesadministering an effective pest-inhibiting amount of a Formula 2compound orally, parenterally, or topically to the animal. In anotherpreferred embodiment, the present invention is directed to a method forinhibiting a susceptible insect of the order Homoptera which comprisesapplying to a plant an effective insect-inactivating amount of a Formula2 compound. Another preferred embodiment of the invention is directed toa method of inhibiting mites of the order Acarina which comprisesapplying to the locus of the mite a mite-inactivating amount of aFormula 2 compound.

Mite/Insect Screen

The Formula 2 compounds were tested for miticidal and insecticidalactivity in the following mite/insect screen. Each test compound wasformulated by dissolving the compound in an acetone-alcohol (1:1)mixture containing 23 g of TOXIMUL R (sulfonate/nonionic emulsifierblend) and 13 g of TOXIMUL S (sulfonate/nonionic emulsifier blend) perliter. These mixtures were then diluted with water to give the indicatedconcentrations.

Two-spotted spider mites and cotton aphids were introduced on squashcotyledons and allowed to establish on both leaf surfaces. The leaveswere then sprayed with 5 ml of test solutions using a DeVilbissatomizing sprayer at 10 psi. Both surfaces of the leaves were covereduntil run off and then allowed to dry for one hour. After standardexposure periods percent mortality was evaluated. Additional insectswere evaluated using similar formulations and evaluation procedures. Theresults are reported in Table IX. The following abbreviations are used:

    ______________________________________                                        Abbreviation                                                                          Pest             Scientific Name                                      ______________________________________                                        ALH     Aster Leafhopper Macrosteles fascifrons                               BAW     Beet Armyworm    Spodoptera exiqua                                    CA      Cotton Aphid     Aphis gossypii Glover                                GECR    German Cockroach Blattella germanica                                  NEM     Rootknot Nematode                                                                              Meliiodyne spp.                                      SCRW    Southern Corn Rootworm                                                                         Diabrotica                                                                    undecimpunctata howardi                              TBW     Tobacco Budworm  Heliothis virescens                                  TSSM    Two-spotted Spider Mite                                                                        Tetranychus urticae                                  ______________________________________                                    

                                      TABLE IX                                    __________________________________________________________________________    Activity of Formula 2 Compounds in Insect/Mite Screen                                    % Inhibition.sup.b                                                 Pest                                                                              rate.sup.a                                                                       per.sup.c                                                                         A83543K                                                                            A83543O                                                                            A83543P                                                                            A83543U                                                                            A83543W                                                                            A83543Y                                                                            N-demethyl-K                         __________________________________________________________________________    ALH 200                                                                              24 hr                                                                             100  100  0    100  0    60   100                                      400                                                                              24 hr                                                                             100  100  0    100  0    80   100                                  BAW 200                                                                              6 day                                                                             100  100  100  100  0    80   100                                      400                                                                              6 day                                                                             100  100  100  100  60   100  100                                  CA  200                                                                              4-5 day                                                                           0    0    0    0    0    0    0                                        400                                                                              4-5 day                                                                           0    100  0    0    0    0    0                                    GECR                                                                              200                                                                              7 day                                                                             0    0    0    0    0    0    0                                        400                                                                              7 day                                                                             0    0    0    0    0    0    0                                        200                                                                              21 day                                                                            0    20   20   0    20   --   20                                       400                                                                              21 day                                                                            80   100  0    0    0    --   60                                   NEM 200                                                                              11 day                                                                            0    0    0    0    0    --   --                                       400                                                                              11 day                                                                            0    0    0    0    0    --   100                                  SCRW                                                                              200                                                                              11 day   0    0    0    0    0    --                                       400                                                                              11 day                                                                            100  0    0    0    0    0    60                                   TBW 200                                                                              6 day                                                                             100  100   0 (50)                                                                            100 (50)                                                                           0 (50)                                                                             0 (50)                                                                             100 (50)                                 400                                                                              6 day                                                                             100  100  100  100  100  100  100                                  TSSM                                                                              200                                                                              4-5 day                                                                           90   100  100 (50)                                                                           0    0 (50)                                                                             0 (50)                                                                              0 (50)                                  400                                                                              4-5 day                                                                           100  100  100  0    80   100  100                                  __________________________________________________________________________     .sup.a rate in ppm (unless otherwise indicated in parenthesis)                .sup.b % inhibition as a mean of single replicate tests                       .sup.c exposure period.                                                  

Formula 2 compounds were evaluated in the following assay to determinethe LD₅₀ against neonate tobacco budworm (Heliothis virescens). A petridish (100 mm×20 mm) is inverted and the lid lined with a #1 qualitativefilter pacer. Ten neonate larvae are placed in each dish and a 1 ml testsolution is pipetted onto the insects. The petri dish bottom is thenplaced on the lid to contain the larvae. At 1 hour after treatment, asmall piece of Heliothis diet (modified slurry, Southland Products, LakeVillage, Ark.) is added to each dish. The mortality is evaluated at 24and 48 hours. The tests were run in triplicate. The results are shown inTable X.

                  TABLE X                                                         ______________________________________                                        Activity Against Neonate Tobacco Budworm                                      Compound       LD.sub.50 (ppm).sup.a                                          ______________________________________                                        A83543K        3.5                                                            A83543O        1.4                                                            A83543P        >64                                                            A83543U        22                                                             A83543W        >64                                                            A83543Y        20                                                             N-de-methyl-K  9.8                                                            ______________________________________                                         .sup.a mean of two tests                                                 

Insecticidal Compositions

The Formula 2 compounds of this invention are applied in the form ofcompositions, which are also a part of this invention. Thesecompositions comprise an insect- or mite-inactivating amount of aFormula 2 compound in a phytologically acceptable inert carrier. Theactive component, the Formula 2 compound, may be present as a singleFormula 2 compound, a mixture of two or more Formula 2 compounds, amixture of at least one of A83543K, A83543O, A83543P, A83543U, A83543V,A83543W and A83543Y or a mixture of at least one of A83543K, A83543O,A83543P, A83543U, A83543V, A83543W and A83543Y together with the driedportion of the fermentation medium in which it is produced.

Compositions are prepared according to procedures and formula which areconventional in the agricultural chemical art, but which are novel andimportant because of the presence of one or more of the compounds ofthis invention. The compositions are either concentrated formulationswhich are dispersed in water for application or dust or granularformulations which are applied without further treatment.

The dispersions in which the compound or crude dried material areapplied are most often aqueous suspensions or emulsions prepared fromconcentrated formulations of the compounds or crude material. Suchwater-soluble, water-suspendible, or emulsifiable formulations areeither solids (usually known as wettable powders) or liquids (usuallyknown as emulsifiable concentrates or aqueous suspensions).

Wettable powders, which may be compacted to form water dispersiblegranules, comprise an intimate mixture of the active compound, an inertcarrier, and surfactants. The concentration of the active compound isusually from about 1% to about 90% by weight. The inert carrier isusually chosen from among attapulgite clays, the montmorillonite clays,the diatomaceous earths or the purified silicates.

Effective surfactants, comprising from about 0.5% to about 10% of thewettable powder are found among the sulfonated lignins, the condensednaphthalene-sulfonates, the napthalene-sulfonates, thealkyl-benzenesulfonates, the alkylsulfates, and nonionic surfactantssuch as ethylene oxide adducts of alkylphenols.

Emulsifiable concentrates of the compounds comprise a convenientconcentration of a compound, such as from about 50 to about 500 gramsper liter of liquid, equivalent to about 10% to about 50%, dissolved inan inert carrier which is either a water-miscible solvent or mixture ofa water-immiscible organic solvent and emulsifiers. Useful organicsolvents include aromatics, especially the xylenes, and petroleumfractions, especially high-boiling naphthlenic and olefinic portions ofpetroleum such as heavy or aromatic naphtha. Other organic solvents mayalso be used, such as the terpenic solvents, including rosinderivatives, aliphatic ketones such as cyclohexanone, and complexalcohols such as 2-ethoxyethanol. Suitable emulsifiers for emulsifiableconcentrates are chosen from conventional nonionic surfactants, such asthose mentioned above.

Aqueous suspensions comprise suspensions of water-insoluble compounds ofthis invention dispersed in an aqueous vehicle at a concentration in therange from about 5% to about 50% by weight. The suspensions are preparedby finely grinding the compound, and vigorously mixing it into a vehiclecomprised of water and surfactants chosen from the same types discussedabove. Inert ingredients, such as inorganic salts and synthetic ornatural gums may also be added to increase the density and viscosity ofthe aqueous vehicle. It is often most effective to grind and mix thecompound at the same time by preparing the aqueous mixture andhomogenizing it in an implement such as a sand mill, ball mill, orpiston-type homogenizer.

The Formula 2 compounds may also be applied as granular compositions,which are particularly useful for applications to the soil. Granularcompositions usually contain from about 0.5% to about 10% by weight ofthe Formula 2 compound, dispersed in an inert carrier which consistsentirely or in large part of clay or a similar inexpensive substance.Such compositions are usually prepared by dissolving the compound in asuitable solvent and applying it to a granular carrier which has beenpre-formed to the appropriate particle size in the range of from about0.5 to 3 mm. Such compositions may also be formulated by making a doughor paste of the carrier, drying the combined mixture of the activeingredient in the dough or paste, and crushing the dried composition toobtain the desired granular particle size.

Dusts containing the compound are prepared by intimately mixing thecompound in powdered form with a suitable dust agricultural carrier,such as kaolin clay, ground volcanic rock, and the like. Dusts cansuitably contain from about 1% to about 10% of the Formula 2 compound.

It is equally practical, when desirable for any reason, to apply thecompound in the form of a solution in an appropriate organic solvent,usually a bland petroleum oil, such as the spray oils, which are widelyused in agricultural chemistry.

Insecticides and miticides are usually applied in the form of adispersion of the active ingredient in a liquid carrier. It isconventional to refer to application rates in terms of the concentrationof active ingredient in the carrier. The most widely used carrier iswater.

The Formula 2 compounds can also be applied in the form of an aerosolcomposition. In such compositions the active compound is dissolved in aninert carrier, which is a pressure-generating propellant mixture. Theaerosol composition is packaged in a container from which the mixture isdispersed through an atomizing valve. Propellant mixtures compriseeither low-boiling halocarbons, which may be mixed with organicsolvents, or aqueous suspensions pressurized with inert gases or gaseoushydrocarbons.

The amount of compound to be applied to the loci of insects and mites isnot critical and can readily be determined by those skilled in the artin view of the examples provided. In general, concentrations of fromabout 10 ppm to about 5,000 ppm of the Formula 2 compound are expectedto provide good control. With many of the compounds, concentrations offrom about 100 to about 1,000 ppm will suffice. For field crops, such assoybeans and cotton, a suitable application rate for the compounds isabout 0.01 to about 1 kg/ha, typically applied in a 5 to 50 gal/A ofspray formulation.

The locus to which a Formula 2 compound is applied can be any locusinhabited by an insect or mite, for example, vegetable crops, fruit andnut trees, grape vines and ornamental plants. Because of the uniqueability of mite eggs to resist toxicant action, repeated applicationsmay be desirable to control newly emerged larvae, as is true of otherknown acaricides.

Ectoparasiticide Activity

The Formula 2 compounds are also active against members of the insectorder Diptera. Tables XI and XII summarize the in vitro studies of theFormula 2 compounds against blowfly larvae and adult stable fly at 48hours.

                  TABLE XI                                                        ______________________________________                                        Activity Against Blowfly Larvae                                                              Activity                                                       Compound       rate (ppm)                                                                             % mortality                                           ______________________________________                                        A83543K        2.5      40                                                    A83543P        10.0     10                                                    A83543W        10.0     0                                                     A83543Y        10.0     100                                                   ______________________________________                                    

                  TABLE XII                                                       ______________________________________                                        Activity Against Adult Stable fly                                                            Activity                                                       Compound       rate (ppm)                                                                             % mortality                                           ______________________________________                                        A83543K        2.5      90                                                    A83543O        5        100                                                   A83543P        10       90                                                    A83543W        10       40                                                    A83543Y        10       90                                                    ______________________________________                                    

Ectoparasiticidal Methods

The ectoparasiticidal method of this invention is carried out byadministering a Formula 2 compound to host animals to control insect andAcarina parasites. Administration to the animal may be by the dermal,oral, or parenteral routes.

Parasitic insects and Acarina include species that are bloodsucking aswell as flesh eating and are parasitic during all of their life cycle oronly part of their life cycle, such as only the larval or only the adultstage. Representative species include the following:

    ______________________________________                                        horse fly         Tabanus spp.                                                stable fly        Stomoxys calcitrans                                         black fly         Simulium spp.                                               horse sucking louse                                                                             Haematopinus asini                                          mange mite        Sarcoptes scabiei                                           scab mite         Psoroptes equi                                              horn fly          Haematobia irritans                                         cattle biting louse                                                                             Bovicola bovis                                              shortnosed cattle louse                                                                         Haematopinus                                                                  eurysternus                                                 longnosed cattle louse                                                                          Linoqnathus vituli                                          tsetse fly        Glossina spp.                                               cattle follicle mite                                                                            Demodex bovis                                               cattle tick       Boophilus microplus and                                                       B. decoloratus                                              Gulf Coast tick   Amblyomma maculatum                                         Lone Star tick    Amblyomma americanum                                        ear tick          Otobius meqnini                                             Rocky Mountain    Dermacentor andersoni                                       wood tick                                                                     screw-worm fly    Cochliomyia hominivorax                                     assassin bug      Reduvius spp.                                               mosquito          Culiseta inornata                                           brown ear tick    Rhipicephalus                                                                 appendiculatus                                              African red tick  Rhipicephalus evertsi                                       bont tick         Amblyomma sp.                                               bont legged tick  Hyalomma sp.                                                hog louse         Haematopinus suis                                           chigoe            Tunqa penetrans                                             body louse        Haematopinus ovillus                                        foot louse        Linoqnathus pedalis                                         sheep ked         Melophaqus ovinus                                           sheep scab mite   Psoroptes ovis                                              qreenbottle fly   Phaenicia sericata                                          black blow fly    Phormia reqina                                              secondary screw-worm                                                                            Cochliomyia macellaria                                      sheep blow fly    Phaenicia cuprina                                           bed bug           Cimex lectularius                                           Southern chicken flea                                                                           Echidnophaqa qallinacea                                     fowl tick         Arqas persicus                                              chicken mite      Dermanyssus qallinae                                        scalyleg mite     Knemidokoptes mutans                                        depluming mite    Knemidokoptes qallinae                                      dog follicle mite Demodex canis                                               dog flea          Ctenocephalis canis                                         American dog tick Dermacentor variabilis                                      brown dog tick    Rhipicephalus                                                                 sanguineus                                                  ______________________________________                                    

The method of the invention may be used to protect economic andcompanion animals from ectoparasites. For example, the compound maybeneficially be administered to horses, cattle, sheep, pigs, goats,dogs, cats and the like, as well as to exotic animals such as camels,llamas, deer and other species which are commonly referred to as wildanimals. The compound may also beneficially be administered to poultryand other birds, such as turkeys, chickens, ducks and the like.Preferably, the method is applied to economic animals, and mostpreferably to cattle and sheep.

Ectoparasiticidal Compositions

This invention also relates to compositions for controlling a populationof insect ectoparasites which consume blood of a host animal. Thesecompositions may be used to protect economic, companion, and wildanimals from ectoparasites. The compositions may also beneficially beadministered to poultry and other birds.

Preferably, the method is applied or the compositions are used toprotect economic animals, and most preferably to cattle and sheep. Therate, timing and manner of effective application will vary widely withthe identity of the parasite, the degree or parasital attack and otherfactors. Applications can be made periodically over the entire life spanof the host, or for only peak season of parasitic attack. In generalectoparasite control is obtained with topical application of liquidformulations containing from about 0.0005 to about 95% of the Formula 2compound, preferably up to 5%, and most preferably up to 1% of a Formula2 compound. Effective parasite control is achieved at an administrationrate from about 5 to about 100 mg/kg.

The Formula 2 compounds are applied to host animals by conventionalveterinary practices. Usually the compounds are formulated intoectoparasiticidal compositions which comprise a Formula 2 compound and aphysiologically-acceptable carrier. For example, liquid compositions maybe simply sprayed on the animals for which ectoparasiticidal control isdesired. The animals may also treat themselves by such devices as backrubbers which may contain the Formula 2 compound and a cloth, forexample, which the animal may walk against in contact. Dip tanks arealso employed to administer the active agent to the host animal.

Oral administration may be performed by mixing the compound in theanimals' feed or drinking water, or by administering dosage forms suchas tablets, capsules, boluses or implants. Percutaneous administrationis conveniently accomplished by subcutaneous, intraperitoneal, andintravenous injection of an injectible formulation.

The Formula 2 compounds can be formulated for oral administration in theusual forms, such as drenches, tablets or capsules. Such compositions,of course, require orally-acceptable inert carriers. The compounds canalso be formulated as an injectible solution or suspension, forsubcutaneous, dermal, intraruminal, intraperitoneal, intramuscular, orintravenous injection. In some applications the compounds areconveniently formulated as one component of a standard animal feed. Inthis embodiment it is usual to formulate the present compound first as apremix in which the compound is dispersed in a liquid or particulatesolid carrier. The premix can contain from about 2 to about 250 g ofFormula 2 compound per pound of mix. The premix is in turn formulatedinto the ultimate feed by conventional mixing.

Because ectoparasitic attack generally takes place during a substantialportion of the host animal's life span, it is preferred to administerFormula 2 compounds in a form to provide sustained release over a periodof time. Conventional procedures include the use of a matrix whichphysically inhabits dissolution, where the matrix is a waxy semi-solid,such as the vegetable waxes, or a high molecular weight polyethyleneglycol. A good way to administer the compounds is by means of asustained-action bolus, such as those of Laby, U.S. Pat. No. 4,251,306and Simpson, British Patent No. 2,059,716. For such a bolus the compoundwould be encapsulated in a polymeric matrix such as that of Nevin, U.S.Pat. No. 4,273,920. Sustained release of the compounds of the presentinvention can also be achieved by the use of an implant such as from asilicone-containing rubber.

In order to illustrate more fully the operation of this invention, thefollowing examples are provided:

EXAMPLE 1

A83543 Assay Method

The following analytical high performance liquid chromatography (HPLC)method is useful for monitoring a fermentation for the production ofA83543K, A83543O, A83543P, A83543U, A83543V, A83543W, A83543Y and otherA83543 components:

A sample of the whole broth is diluted with three volumes ofacetonitrile to extract the factors from the mycelia. The resultingsolution is then filtered through a 0.45 micron polytetrafluorine (PTFE)filter to remove particulate matter prior to injection into the HPLCassay system. A solution of purified A83543A at a concentration of 100mg/ml in methanol is used as an external standard for the assay and peakareas of all A83543 components are related back to this calibrationstandard to determine concentrations of individual components.

HPLC System:

Column Support: YMC-PACK 4.6×100-mm ID column, 5μ spherical, 120Å (YMCInc., Morris Plains, N.J.)

Mobile Phase: CH₃ CN/MeOH/H₂ O (3:3:2) containing

    ______________________________________                                        Flow Rate:   2 ml/min                                                         Detection:   UV at 250 nm                                                     Retention Times:                                                                           A83543A         15.52  min                                                    A83543K         8.10   min                                                    A83543O         11.40  min                                                    A83543P         6.40   min                                                    A83543U         5.22   min                                                    A83543V         7.05   min                                                    A83543W         8.47   min                                                    A83543Y         6.12   min                                       ______________________________________                                    

EXAMPLE 2

Preparation of A83543K and A83543O with Culture NRRL 18538 (A83543.4)

A. Shake-flask Fermentation

The culture S. spinosa NRRL 18538, either as a lyophilized pellet or asa suspension maintained in liquid nitrogen, was used to inoculate avegetative medium having the following composition:

    ______________________________________                                        Vegetative Medium 1                                                           Ingredient              Amount (g)                                            ______________________________________                                        Enzyme-hydrolyzed casein*                                                                             30                                                    Yeast extract           3                                                     MgSO.sub.4 · 7H.sub.2 O                                                                      2                                                     Glucose                 10                                                    Deionized water         q.s. 1-L                                              pH 6.2, adjust to pH 6.5 with NaOH                                            ______________________________________                                         *NZ Amine A, Sheffield Products, Norwich, NY.                            

Slants or plates can be prepared by adding 2.5% agar to the vegetativemedium. The inoculated slant is incubated at 30° C. for about 10 toabout 14 days. The mature slant culture is scraped with a sterile toolto loosen the spores and to remove and macerate the mycelial mat. Aboutone-fourth of the loosened spores and culture growth thus obtained isused to inoculate 50 ml of a first-stage vegetative medium.Alternatively, the first-stage medium may be inoculated from a liquidnitrogen ampoule.

When culture is maintained in liquid nitrogen, ampoules are preparedusing equal volumes of vegetative culture (48-72 hours incubation, 30°C.) and suspending medium. The suspending medium contains lactose (100g), glycerol (200 ml), and deionized water (q.s. to l-L).

A liquid nitrogen ampoule is used to inoculate 50 ml of vegetativemedium in 250-ml Erlenmeyer flasks. The cultures are incubated at 30° C.for 48 hours on a shaker orbiting in a two-inch (5.08 cm) circle at 250rpm.

The incubated culture (5% v/v inoculum) is used to inoculate 30 ml of aproduction medium in a 250-ml wide-mouth Erlenmeyer flask. The mediumcomposition was as follows:

    ______________________________________                                        Production Medium                                                             Ingredient            Amount (g)                                              ______________________________________                                        Glucose               80                                                      Peptonized milk*      20                                                      Cottonseed flour**    30                                                      Corn steep liquor     10                                                      CaCO.sub.3 (tech. grade)                                                                             5                                                      Methyl oleate         30***                                                   Tap water             q.s. to 1-L                                             ______________________________________                                         *Peptonized Milk Nutrient, Sheffield Products, Norwich, NY                    **Proflo, Traders Protein, Memphis, TN                                        ***The amount of methyl oleate was 30 ml                                 

The inoculated production medium is incubated in 250-ml wide-mouthErlenmeyer flasks at 30° C. for 7 days on a shaker orbiting in atwo-inch circle at 250 rpm. Sinefungin was added at a finalconcentration of about 100 μg/ml, at 72 hours after inoculation.

B. Stirred Reactor Fermentation

In order to provide a larger volume of inoculum, 10 ml of incubatedfirst stage medium, prepared as described in Example 2, Section A, isused to inoculate 400 ml of a second-stage vegetative medium having thesame composition as that of the first-stage medium. This second-stagevegetative medium is incubated in a 2-L wide-mouth Erlenmeyer flask forabout 48 hours at 30° C. on a shaker orbiting in a two-inch circle at250 rpm.

Incubated second-stage vegetative medium (2-L) thus prepared is used toinoculate 115 liters of sterile production medium, prepared as describedin Example 2, Section A. Sinefungin, as a filtered methanolic solution,was added at 66 hours to a final concentration of 100 μg/ml.

The inoculated production medium was allowed to ferment in a 165-Lstirred bioreactor for 7 days at a temperature of 30° C. The air-flowand agitator speed in the stirred vessel are computer controlled tomaintain a dissolved oxygen level at about 80% of air saturation.

EXAMPLE 3

Preparation of A83543K, A83543O and A83543Y with Culture NRRL 18743(A83543.8)

A. Shake-flask Fermentation

The culture S. spinosa NRRL 18743, either as a lyophilized pellet or asa suspension maintained in liquid nitrogen, was used to inoculate avegetative medium having the following composition:

    ______________________________________                                        Vegetative Medium 2                                                           Ingredient        Amount (g)                                                  ______________________________________                                        Trypticase soy broth*                                                                           30                                                          Yeast extract     3                                                           MgSO.sub.4 · 7H.sub.2 O                                                                2                                                           Glucose           5                                                           Maltose           4                                                           Deonized water    q.s. 1-L                                                    autoclave 30 min at 120° C.                                            ______________________________________                                         *Baltimore Biological Laboratories, Cockeysville, MD                     

Slants or plates can be prepared by adding 2.5% agar to the vegetativemedium. The inoculated slant is incubated at 30° C. for about 10 toabout 14 days. The mature slant culture is scraped with a sterile toolto loosen the spores and remove and macerate the mycelial mat. Aboutone-fourth of the loosened spores and culture growth thus obtained isused to inoculate 50 ml of a first-stage vegetative medium.Alternatively, the first-stage medium may be inoculated from a liquidnitrogen ampoule.

Liquid-nitrogen-stock inoculum was prepared by homogenizing a vegetativeculture, diluting 1:1 (volume:volume) with a sterile suspending agent ofglycerol:lactose:water (2:1:7), and dispensing into sterile tubes (1.5ml/tube). The diluted inoculum was then stored over liquid nitrogen inappropriate storage containers and used as a working stock inoculum forthe cultivation of shake-flask cultures and fermenter seed inoculum.

A liquid nitrogen ampoule was quick thawed and 0.5 ml was used toinoculate 50 ml of vegetative medium in 250-ml wide-mouth Erlenmeyerflasks. The cultures are incubated at 32° C. for 48 hours on a shakerorbiting in a two-inch (5.08 cm) circle at 250 rpm.

The incubated culture (5% v/v inoculum) is used to inoculate 25 ml of aproduction medium having the following composition:

    ______________________________________                                        Production Medium                                                             Ingredient      Amount (g)                                                    ______________________________________                                        Glucose         80                                                            Peptonized milk*                                                                              20                                                            Cottonseed flour**                                                                            30                                                            Corn steep liquor                                                                             10                                                            CaCO.sub.3 (tech. grade)                                                                       5                                                            Methyl oleate   30                                                            Tap water       q.s. to 1-L                                                   ______________________________________                                         *Peptonized Milk Nutrient, Sheffield Products, Norwich, NY                    **Proflo, Traders Protein, Memphis TN                                    

The inoculated production medium is incubated in 250-ml wide-mouthErlenmeyer flasks at 30° C. for 7 days on a shaker orbiting in atwo-inch circle at 250 rpm.

B. Stirred Reactor Fermentation

In order to provide a larger volume of inoculum, 10 ml of incubatedfirst stage medium, prepared as described in Example 3, Section A, isused to inoculate 400 ml of a second-stage vegetative medium having thesame composition as that of the first-stage medium. This second-stagevegetative medium is incubated in a 2-L wide-mouth Erlenmeyer flask forabout 48 hours at 32° C. on a shaker orbiting in a two-inch circle at250 rpm.

Incubated second-stage vegetative medium (2-L) thus prepared is used toinoculate 115 liters or sterile production medium, prepared as describedin Example 3, Section A.

The inoculated production medium is allowed to ferment in a 165-Lstirred bioreactor for 7 days at a temperature of 30° C. The air-flowand agitator speed in the stirred vessel are computer controlled tomaintain a dissolved oxygen level at or above 80% of air saturation.

EXAMPLE 4

Isolation of A83543P and A83543W from NRRL 18719 (A83543.6) fermented inthe presence of sinefungin

Fermentation broth (190-L stirred fermenter), prepared substantially asdescribed in Example 2B (with the exception that strain A83543.6 wasused), was refrigerated two days prior to processing. Acetone (190-L)was added to the whole broth after adjusting thepH to 3.0 with 5N HCl.The resulting mixture was filtered through a ceramic filter to givefiltrate (335-L) which was held over the weekend under refrigeration.The broth/acetone filtrate was adjusted to pH 10 with 5N NaOH andrefiltered through the ceramic filter prior to loading onto a steelcolumn (10-L; 10 cm×122 cm) containing HP-20ss resin (MitsubishiChemical Industries, Ltd., Japan) at a flow rate of 1-L/minute. Thecolumn was washed with CH₃ CN--CH₃ OH-- 0.1% aq. NH₄ OAc (adjusted to pH8.1 with NH₄ OH) (25:25:50: 20-L). then eluted with CH3CN--CH₃ OH--0.1%aq. NH₄ OAc (adjusted pH 8.1 with NH₄ OH) (95:95:10; 40-L), collecting2-L fractions. Fractions 3-9 were concentrated to dryness, redissolvedin CH₃ OH (100 ml), reconcentrated, then precipitated into CH₃ CN (1-L).The resulting precipitate was removed by filtration and discarded; thefiltrate was concentrated to dryness. The resulting residue wasredissolved in dichloromethane (25 ml) and applied to a column (7.5cm×50 cm) of silica gel (EM grade 62, 60-200 mesh) equilibrated inacetonitrile. The column was eluted with CH₃ CN (4-L), then CH₃ CN--CH₃OH (9:1; 5-L), followed by CH₃ OH (1-L), collecting 1-L fractions. Pool1 (fractions 3-4) contained A83543 components J and L; pool 3 (fractions7-10), components M and N. Pool 2 (fractions 5-6), containing newcomponents P and W, was concentrated to dryness. The resulting residuewas dissolved in CH₃ OH (10 ml) and applied to a preparative reversephase HPLC column (Rainin Dynamax-60Å 8 μm C18, 41.4 mm ID×25 cm with41.4 mm×5 cm guard module) equilibrated in H₂ O--CH₃ OH--CH₃ CN;(30:35:35, containing 0.1% NH₄ OAc). The column was eluted at a flowrate of 40 ml/minute with a gradient mixed from solvent "A" H₂ O--CH₃OH--CH₃ CN (30:35:35, containing 0.1% NH₄ OAc) and solvent "B" H₂ O--CH₃OH--CH₃ CN; (10:45:45, containing 0.1% NH₄ OAc). The pumping system wasprogrammed to generate a linear gradient from 25 to 75% B in 60 minutes.Progress of the separation was monitored with a variable wavelength UVdetector tuned to 250 nm. The major peak was collected in 6×3 minutefractions. Fractions 1-2, containing new component P, were concentratedto 40 ml, then desalted on the same HPLC column equilibrated in H₂O--CH₃ OH--CH₃ CN (30:35:35) by eluting with a 60 minute linear gradientfrom H₂ O--CH₃ OH--CH₃ CN (30:35:35) to H₂ O--CH₃ OH--CH₃ CN (10:45:45).The UV absorbing peak (minus the first 2 minutes eluted) was collectedand concentrated to dryness. The resulting residue was dissolved int-BuOH (10 ml) and lyophilized to give pure component P (479 mg). Pooledfractions 3-4 from above, containing a mixture of component P and W,were concentrated to 20 ml and applied to a preparative reverse phaseHPLC column (Rainin Dynamax-60Å 8 μm C18, 21.4 mm ID×25 cm with 21.4mm×5 cm guard module), equilibrated in H₂ O--CH₃ OH--CH₃ CN (30:35:35)containing 0.1% NH₄ OAc, and eluted at a flow rate of 10 ml/minute witha gradient mixed from solvent "A" H₂ O--CH₃ OH--CH₃ CN; (30:35:35,containing 0.1% NH₄ OAc) and solvent "B" H₂ O--CH₃ OH--CH₃ CN;(10:45:45, containing 0.1% NH₄ OAc). The pumping system was programmedto generate a linear gradient from 25 to 75% B in 60 minutes. Two majorUV absorbing peaks (component P, followed by component W) werecollected. The component W containing pool was concentrated to a smallvolume, then desalted on the same HPLC column equilibrated in H₂ O--CH₃OH--CH₃ CN (30:35:35). Component W was eluted with a 60 minute lineargradient from H₂ O--CH₃ OH--CH₃ CN (30:35:35) to H₂ O--CH₃ OH--CH₃ CN(10:45:45) at a flow rate of 10 ml/minute, collecting UV absorbing peakinto 10×3 minute fractions. Pooled fractions 2-7 were concentrated toresidue, dissolved in t-BuOH, and lyophilized to give pure component W(82 mg). The component P-containing UV absorbing peak from above wasdesalted in like manner to give additional pure component P (132 mg).

EXAMPLE 5

Isolation of A83543U and A83543V from strain NRRL 18823 (A83543.9)fermented in the presence of sinefungin

Fermentation broth (500 ml; 30×250 ml shake flasks) preparedsubstantially as described in Example 2A (except strain A83543.9 wasused), was extracted with methanol (1.3-L) with stirring for one hour,then filtered using a filter aid (3% Hyflo) to give methanolic filtrate(1.5-L). The biomass was reextracted with methanol (700 ml) andfiltered. The two methanolic extracts were combined and an equal volumeof water added. HP-20 resin (75 ml) was added and stirred for 2 hours,after which the slurry was poured into a glass chromatography column.The effluent (5-L) was discarded, as was a CH₃ OH--H₂ O (1:1) wash (500ml) of the column. The column was then eluted with acetone (250 ml). Theacetone eluate was combined with that obtained from a similar extractionand chromatography of whole broth (500 ml; 40×250 ml shake flasks) andconcentrated to dryness. The resulting residue was dissolved indichloromethane (10 ml) and applied to a column (2.5 cm×25 cm) of silicagel (EM grade 62, 60-200 mesh) equilibrated in acetonitrile. The columnwas washed with acetonitrile, then eluted with a linear gradient fromacetonitrile to acetonitrile-methanol (4:1), collecting 25 ml fractions.Fractions 34-43, containing new A83543 components U and V were pooled(200 ml), and concentrated to dryness. The residue was dissolved inmethanol (2 ml) and applied to a preparative reverse phase HPLC column(Rainin Dynamax-60Å 8 μm C18, 21.4 mm ID×25 cm with 21.4 mm×5 cm guardmodule) equilibrated in H₂ O--CH₃ OH--CH₃ CN (30:35:35) containing 0.1%NH₄ OAc. The column was eluted with a 60 minute linear gradient from H₂O--CH₃ OH --CH₃ CN (30:35:35) containing 0.1% NH₄ OAc to H₂ O--CH₃ OH--CH₃ CN (10:45:45) containing 0.1% NH₄ OAc at a flow rate of 10ml/minute. The major peaks (UV monitored at 250 nm), containing newcomponents U and V, were collected before residual components H and Q.The pool containing component U was desalted on the same HPLC columnequilibrated in H₂ O--CH₃ OH--CH₃ CN (30:35:35) by eluting with a lineargradient from H₂ O--CH₃ OH--CH₃ CN (30:35:35) to H₂ O--CH₃ OH--CH₃ CN(10:45:45). Component U was eluted in 2 minute fractions (10). Fractions2-8 were pooled, then concentrated to dryness. The residue was dissolvedin t-BuOH (5 ml) and lyophilized to give pure component U (71 mg). Thecomponent V-containing pool was desalted and lyophilized by the sameprocedure to give pure component V (7 mg).

EXAMPLE 6

Isolation of A83543K and A83543O from NRRL 18538 (A83543.4) fermented inthe presence of sinefungin

Fermentation broth (210-L stirred fermenter) was prepared substantiallyas described in Example 2B. Acetone was added to the whole broth and thepH was adjusted to 8.0. The resulting mixture was filtered through aceramic filter to give filtrate (370-L). The broth/acetone filtrate wasloaded onto a steel column (10-L, 10 cm×122 cm) containing HP-20ss resin(Mitsubishi Chemical Industries, Ltd., Japan) at a flow rate of1-L/minute, collecting the effluent an a single pool. The column waseluted at a flow rate of 1-L/minute with a gradient mixed from solvent"A" (0.1% NH₄ OAc) and solvent "B" (CH₃ OH--CH₃ CN; 1:1). The pumpingsystem was programmed to deliver 50% B for 2 minutes, followed by alinear gradient from 50-80B (45 minutes), followed by a linear gradientfrom 80-90% B (33 minutes), collecting 20×4 L fractions. Fractions13-17, containing components K and O were pooled. The column effluent(see above) was adjusted to pH 9.5 with 5N NaOH and reapplied to theHP-20ss column. The pumping system was programmed to deliver 50% B for 1minute, a linear gradient from 50-75% B (30 minutes), a linear gradientfrom 75-85% B (45 minutes), a linear gradient from 85-88% B (15.4minutes), and a linear gradient from 88-100% B (20 minutes), at a flowrate of 1-L/minute, collecting 22×4 L fractions. Fractions 7-17 werepooled and combined with the pool (fractions 13-17 from the firstHP-20ss chromatography (see above). The combined pools were concentratedto 4-L, then further concentrated to dryness, redissolved in CH₃ OH (100ml), then precipitated into CH₃ CN (3-L). The resulting precipitate wasremoved by filtration, washed with CH₃ CN, and discarded; the filtratewas concentrated to dryness. The resulting residue was redissolved indichloromethane (50 ml) and applied to a column (6 cm ×24 cm) of silicagel (EM grade 62, 60-200 mesh) equilibrated in acetonitrile. The columnwas eluted with CH₃ CN (4-L), then CH₃ CN--CH₃ OH (9:1; 10-L), taking10×250 ml fractions, followed by 7×1 L fractions. Fractions 6-15,containing components K and O, were concentrated to dryness. Theresulting residue was dissolved in CH₃ OH (100 ml) and applied (in 20runs) to a preparative reverse phase HPLC column (Rainin Dynamax-60Å 8μm C18, 41.4 mm ID×25 cm with 41.4 mm×5 cm guard module) equilibrated inH₂ O--CH₃ OH--CH₃ CN; (50:175:175, containing 0.1% NH₄ OAc). The columnwas eluted at a flow rate of 40 ml/minute. Progress of the separationwas monitored with a variable wavelength UV detector tuned to 250 nm. UVabsorbing peaks (from the 20 chromatographic runs) were collected in 7pools. The two largest peaks corresponded to components K and O. Pool 3(6-L), contained component K (98% pure). Pool 4 (8-L), containingcomponents O and K, was concentrated to 200 ml and rechromatographed (in4 runs) under the same conditions, collecting the two peaks as twopools. Pool 1 (3-L) contained component K (98% pure). Pool 2 (5-L),contained component O (95%) and component K (5%). Pool 2 wasconcentrated to 100 ml and desalted by chromatography on the same HPLCcolumn (in 3 runs), eluting with a 60 minute linear gradient from H₂O--CH₃ OH--CH₃ CN (30:35:35) to H₂ O--CH₃ OH--CH₃ CN (10:45:45). The UVabsorbing eluate was collected in 10×3 minute fractions. Fractionscontaining >98% pure component O were pooled, concentrated to dryness,and lyophilized from t-BuOH to give component O (2.5 g; >98% pure).Component K containing pools from the first preparative HPLC separation(pool 3, 6-L) and the repurification of component O (pool 1, 3-L) werecombined, concentrated to 200 ml, and desalted in the same manner ascomponent O. Fractions containing >98% pure component K were pooled,concentrated to dryness, and lyophilized from t-BuOH to give component K(11.1 g; >99% pure).

EXAMPLE 7

Isolation of A83543K, A835430, and A83543Y from strain NRRL 18743(A83543.8)

Fermentation broth (260-L) was prepared as substantially described inExample 3B. Acetone (260-L) was added to the whole broth after adjustingthe pH to 3.0 with 5N HCl. The resulting mixture was filtered through aceramic filter to give filtrate (480-L) which was held over the weekendunder refrigeration. The broth/acetone filtrate was adjusted to pH 12with 25% NaOH and refiltered twice through the ceramic filter prior toloading onto a steel column (10-L, 10 cm×122 cm) containing HP-20ssresin (Mitsubishi Chemical Industries, Ltd., Japan) at a flow rate of0.5-L/minute. The column was washed with CH₃ CN--CH₃ OH-- 0.1% aq. NH₄OAc (adjusted to pH 8.1 with NH₄ OH) (25:25:50; 20-L). New components K,O and Y were eluted with CH₃ CN--CH₃ OH -- 0.1% aq. NH₄ OAc (adjusted topH 8.1 with NH₄ OH) (95:95:10; 30-L) at a flow rate of 1-L/minute. Theeluate (30-L) was concentrated, redissolved in CH₃ OH, reconcentrated todryness, redissolved in CH₃ OH (100 ml), then precipitated into CH3CN(2-L). The resulting precipitate was removed by filtration, washed withCH₃ CN, and discarded: the combined filtrate and wash (3-L) wasconcentrated to dryness. The resulting residue was redissolved indichloromethane (50 ml) and applied to a column (7.5 cm×50 cm) of silicagel (EM grade 62, 60-200 mesh) equilibrated in acetonitrile. The columnwas eluted with CH₃ CN (10-L), then CH₃ CN--CH₃ OH (9:1; 20-L), followedby CH₃ CN--CH₃ OH (8:2; 10-L), collecting 1-L fractions. Fractions 11-30were pooled and concentrated to dryness. The resulting residue wasdissolved in CH₃ OH (50 ml) and applied (in 10 runs) to a preparativereverse phase HPLC column (Rainin Dynamax-60Å 8 μm C18, 41.4 mm ID×25 cmwith 41.4 mm×5 cm guard module) equilibrated in H₂ O--CH₃ OH --CH₃ CN;(50:175:175, containing 0.1% NH₄ OAc). The column was eluted at a flowrate of 40 ml/minute with a 60 minute linear gradient from H₂ O--CH₃OH--CH₃ CN; (50:175:175, containing 0.1% NH₄ OAc) to H₂ O--CH₃ OH--CH₃CN; (10:45:45, containing 0.1% NH₄ OAc). Progress of the separation wasmonitored with a variable wavelength UV detector tuned to 250 nm. Thefirst three peaks collected (10 runs pooled) corresponded to the elutionof minor component Y (pool 1, 1-L), component K (pool 2, 8-L) andcomponent O (pool 3, 4-L). Pool 2 was concentrated to a small volume,then desalted by rechromatographing on the same column, eluting withoutbuffer. The effluent corresponding to the UV absorption peak wasconcentrated to dryness, dissolved in t-BuOH, and lyophilized to givepure component K (7.3 g). Pool 3 was desalted and lyophilized in likemanner to give pure component O (1.4 g). Pool 1 was desalted by similarchromatography (Rainin Dynamax-60Å 8 μm C18 column, 21.4 mm ID×25 cmwith 21.4 mm×5 cm guard module) and lyophilized in like manner to givepure component Y (46 mg).

EXAMPLE 8

A83543K pseudoaglycone

A sample or A83543K (100 mg) was dissolved 2N sulfuric acid (10 ml).This solution was heated at about 80° C. for 1.25 hours, and theresulting mixture was allowed to cool to room temperature. Theprecipitate was collected by filtration, washed with cold deionizedwater, and dried to give 59 mg of A83543K pseudoaglycone.

Elemental Analysis

MS (FD): m/z 576 (100%)

IR (CHCl₃): 2936.0, 1714.9, 1659.0 cm⁻¹

UV (EtOH): λ_(max) 243 nm

EXAMPLE 9

A83543O Pseudoaglycone

A sample of A83543O (500 mg) was suspended in deionized water (40 ml)and a sufficient volume of lN H₂ SO₄ was added to cause completedissolution (approximately 0.25 ml). The resulting solution was heatedat about 80° C. for 3 hours, and then allowed to cool to roomtemperature. The precipitate was collected by filtration, washed withcold deionized water, and dried. The filtrate was saturated with NaCland extracted with methylene chloride. The methylene chloride extractswere combined, extracted with brine, dried (K₂ CO₃), and evaporated todryness. The residue was combined with the precipitate to give 348 mg ofcrude product.

The crude product was purified by flash chromatography (Silica gel 60,230-400 mesh), eluting with a mixture of ethyl acetate and hexane (7:3).The fractions containing the desired compound were evaporated to drynessto give 146.5 mg of A83543O pseudoaglycone.

Elemental Analysis

MS (FD): m/z 590 (100%), 591 (70%, M+), 592 (20%, M+H), 593 (5&, M+2)

IR (CHCl₃): 3014.2, 2932.2, 1714.9, 1659.0 cm⁻¹

UV (EtOH): λ_(max) 242 nm (ε 9,185)

EXAMPLE 10

N-demethyl-A83543K

A83543K (101.5 mg, 0.14 mmol) and sodium acetate trihydrate (142.4 mg,1.05 mmol) were added to a mixture of methanol and pH 9 buffer solution(Fisher Scientific, Lexington, Mass.). The resulting suspension washeated to about 47° C., and then iodine (47.7 mg, 0.19 mmol) was addedin one portion. After 21/2 hours at 47° C., the reaction was allowed tocool to room temperature. After stirring an additional 3 hours at roomtemperature, the reaction solution was added to a 5% sodium thiosulfatesolution. The resulting colorless aqueous mixture was extracted withdiethyl ether. The aqueous layer was then saturated with NaCl andextracted with methylene chloride. The methylene chloride extracts werecombined with the diethylene extracts, washed with brine, and dried overK₂ CO₃. The dried solution was then evaporated to dryness in vacuo togive 79.3 mg of N-demethyl-A83543K as a white glass (81% yield).

MS (FD) m/z 703 (100%, M+), 704 (57%, m+H), 705 (19%, m+2)

Elemental Analysis (C₃₉ H₆₁ NO₁₀) Calc.: C, 66.55; H, 8.73; N, 99;Found: C, 64.80; H, 8.67; N, 1.95

IR (KBr): 3462.7, 2934.1, 1721.7, 1660.9, 1457.4 cm₋₁.

EXAMPLE 11

di-N-demethyl-A83543K

A solution of N-demethyl-A83543K (891 mg, 1.27 mmol) in MeOH (40 ml) wascooled to 3° C. Freshly prepared lM NaOMe in methanol (6.3 ml, 6.3 mmol)and iodine (1.61 g, 6.3 mmol) were successively added to this solution.The reaction solution was kept at 3° C. for 5 hours, then added to a 5%sodium thiosulfate/-dilute ammonium hydroxide solution. The resultingmixture was extracted with ethyl acetate. The combined ethyl acetateextracts were washed with brine and dried over K₂ CO₃. The driedsolution was evaporated to dryness in vacuo to give 770 mg of crudeproduct.

The desired compound was partially purified by flash chromatography(Silica gel 60, 230-400 mesh, 2 in.×8 in.), eluting with a mixture ofmethylene chloride and methanol (93:7). The desired compound was furtherpurified by reverse-phase HPLC (Waters Prep NOVA-Pak, ODS, 60Å, 40mm×300 mm), eluting with methanol/-acetonitrile/0.25% ammonium acetate(40:40:20). The fractions containing the desired compound were combinedand evaporated to dryness to give 463.6 mg (53% yield) ofdi-N-demethyl-A83543K as a colorless class.

Elemental analysis (C₃₈ H₅₉ NO₁₀) Calc.: C, 66.16; H, 8.62; N, 2.03;Found: C, 66.29; H, 8.63; N, 2.02,

MS(FD): m/z 690 (100%, M+), 689 (70%), 691 (59%, M+H), 704 (20%)

UV (EtOH): λ_(max) 244 nm (ε 10,328)

IR (CHCl₃): 3700, 3600, 3550-3350 (br), 3420, 2975, 1700, 1675, 1620cm₋₁.

What is claimed is:
 1. A compound of the formula 1 ##STR20## wherein R⁷is hydrogen or a group of formula ##STR21## R⁸, R⁹, R¹⁰, R¹¹, and R¹²are independently hydrogen or methyl, provided that R¹¹ and R¹² are notconcurrently hydrogen; or an acid addition salt thereof when R⁷ is otherthan hydrogen.
 2. The compound of claim 1 wherein R⁷ is a group offormula ##STR22##
 3. The compound of claim 2 wherein R⁷ is a group offormula ##STR23##
 4. The compound of claim 3 wherein R⁸ is methyl. 5.The compound of claim 1 wherein R⁷ is hydrogen.
 6. A compound of claim1, wherein R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are for each component asfollows:

    ______________________________________                                        Component R.sup.7                                                                             R.sup.8  R.sup.9                                                                            R.sup.10                                                                             R.sup.11                                                                           R.sup.12                            ______________________________________                                        K         (a)   CH.sub.3 H    CH.sub.3                                                                             CH.sub.3                                                                           CH.sub.3                            O         (a)   CH.sub.3 CH.sub.3                                                                           CH.sub.3                                                                             CH.sub.3                                                                           CH.sub.3                            P         (a)   CH.sub.3 H    CH.sub.3                                                                             CH.sub.3                                                                           H                                   U         (a)   CH.sub.3 H    CH.sub.3                                                                             H    CH.sub.3                            V         (a)   CH.sub.3 CH.sub.3                                                                           CH.sub.3                                                                             H    CH.sub.3                            W         (a)   CH.sub.3 CH.sub.3                                                                           CH.sub.3                                                                             CH.sub.3                                                                           H                                   Y         (a)   CH.sub.3 H    H      CH.sub.3                                                                           CH.sub.3                            ______________________________________                                    


7. An insecticide or miticide composition comprising an insect- ormite-inactivating amount of a compound of claim 2 in combination with aphytologically-acceptable carrier.
 8. An insecticide or miticide methodwhich comprises applying to the locus of an insect or mite an insect- ormite-inactivating amount of a compound of claim
 2. 9. Anectoparasiticidal composition comprising a physiologically-acceptableinert carrier and a compound of claim 2.